A process for extracting high quality dietary fiber from wheat bran

Abstract

This invention relates to a process for extracting high-quality dietary fiber from wheat bran, belonging to the field of wheat by-product processing technology. This invention obtains soluble and insoluble dietary fiber from wheat through processes such as ultrafine grinding, Trichoderma fermentation, enzymatic hydrolysis, and decolorization. The prepared dietary fiber has higher purity and better functional properties. The purity of soluble dietary fiber is 37.1%, and the purity of insoluble dietary fiber is 67.3%. Compared with ordinary enzymatic hydrolysis processes, the purity of soluble dietary fiber is increased by 3.2 times, the purity of insoluble dietary fiber is increased by 54.7%, whiteness is increased by 30-50%, DPPH and FRAP antioxidant properties are increased by 1-2 times, enzyme inhibition is increased by 0.5-2 times, nitrite adsorption capacity is increased by 1.5-1.7 times, and cholesterol adsorption capacity is increased by 3-4 times.

Description

TECHNICAL FIELD

[0001] The present application relates to a process for extracting high-quality dietary fiber from wheat bran, belonging to the technical field of wheat by-product processing. BACKGROUND

[0002] Wheat bran is the main by-product in the process of processing flour from wheat, accounting for about 20% of the weight of the wheat kernel. The main components of wheat bran are carbohydrates (starch and non-starch polysaccharides), proteins, oils, minerals, and vitamins, among which the starch content is 10-15%, and the protein content is 12-18%. The type, origin, and flour processing technology all have an impact on the content of the basic components in the wheat. Non-starch polysaccharides, also known as cell wall polysaccharides, mainly include dietary fiber, accounting for about 46% of the total amount of wheat, of which arabinoxylan accounts for about 70% of the polysaccharide content.

[0003] Currently, dietary fiber is considered to be a general term for large molecules mainly composed of polysaccharides that are not easily digested and absorbed by the human body, including plant pectin, lignin, cellulose, methylcellulose, and animal chitin, collagen, etc. Dietary fiber can be divided into soluble dietary fiber and insoluble dietary fiber according to their water solubility. The main components of insoluble dietary fiber are cellulose, hemicellulose, and lignin, while the main components of soluble dietary fiber are pectin and gum. Dietary fiber has physical effects such as adhesion, water absorption and swelling, ion exchange, and mesh adsorption, and also has effects such as regulating the composition of intestinal microbial flora, reducing blood sugar levels in the human body, lowering blood lipids, and antioxidant effects. Studies have shown that among dietary fiber, insoluble dietary fiber mainly functions to produce mechanical peristalsis in the intestines to accelerate intestinal emptying, while soluble dietary fiber mainly functions to play a metabolic role, such as participating in and affecting carbohydrate and lipid metabolism. Therefore, the proportion of the two components in dietary fiber is an important factor affecting the physiological function of dietary fiber. Some scholars suggest that high-quality dietary fiber should meet the requirement that the content of soluble dietary fiber in the composition of dietary fiber should be more than 10%. In the composition of wheat bran, the proportion of soluble dietary fiber is only 3%-4%, which is far from meeting the requirements of high-quality dietary fiber. SUMMARY

[0004] TECHNICAL PROBLEM

[0005] A process for extracting high-quality dietary fiber from wheat bran is provided, yielding high-quality dietary fiber with a purity of up to 37.1% for soluble dietary fiber and up to 67.3% for insoluble dietary fiber. Compared with traditional enzymatic hydrolysis processes, the purity of soluble dietary fiber is increased by 3.2 times, the purity of insoluble dietary fiber is increased by 54.7%, whiteness is increased by 30-50%, and the functionality of dietary fiber is superior—DPPH and FRAP antioxidant properties are increased by 1-2 times, enzyme inhibition is increased by 0.5-2 times, nitrite adsorption capacity is increased by 1.5-1.7 times, and cholesterol adsorption capacity is increased by 3-4 times.

[0006] Technical solution:

[0007] On the one hand, a process for extracting high-quality dietary fiber from wheat bran is provided, which includes the following steps:

[0008] (1) Pretreatment of wheat bran: Wheat bran is pre-crushed, ultra-fine crushed, and sieved by air classifier to obtain wheat bran powder;

[0009] (2) Preparation of wheat bran liquid culture medium: Mix wheat bran powder, potassium dihydrogen phosphate, magnesium sulfate, ammonium sulfate and calcium chloride, add deionized water, mix well, adjust pH to 3.5, sterilize to obtain wheat bran liquid culture medium; the wheat bran liquid culture medium contains 100mL water, 9-10g wheat bran powder, 0.6-0.7g potassium dihydrogen phosphate, 0.2-0.3g magnesium sulfate, 0.5-0.6g ammonium sulfate and 0.1-0.2g calcium chloride;

[0010] (3) Inoculate the *Trichoderma viride* broth into wheat bran liquid culture medium at an inoculation rate of 10% by volume, shake well, and incubate in a constant temperature incubator at 26-30℃ for 72 hours to obtain the fermentation broth; wherein, the preparation method of the *Trichoderma viride* broth includes: dissolving *Trichoderma viride* spores (strain number: GDMCC NO.3.443) in sterile water, with a *Trichoderma viride* spore concentration of 10... 7 ~10 8 CFU / mL Trichoderma viride spore suspension; add 1 mL of Trichoderma viride spore suspension to 300 mL of liquid culture medium, and culture at 26-30℃ with shaking at 180-200 r / min for 72-80 h to obtain Trichoderma viride solution;

[0011] (4) Sterilize the fermentation broth; then, enzymatically hydrolyze it sequentially using high-temperature α-amylase and neutral protease; then, perform alkaline hydrolysis.

[0012] (5) Add 2.5% by volume of 30% hydrogen peroxide solution, decolorize in a water bath at 35°C for 2.5 h, filter, and obtain filtrate and precipitate;

[0013] (6) Extract water-soluble dietary fiber from the filtrate; extract water-insoluble dietary fiber from the precipitate.

[0014] In some embodiments, in step (1), the mesh size of the ultrafine pulverizer is 120-140 mesh; the mesh size of the airflow sieve is 120 mesh.

[0015] In some embodiments, in step (1), the sterilization conditions in steps (2) and (4) are 121°C, 0.1 MPa, and 15-20 min.

[0016] In some embodiments, the method for preparing the *Trichoderma viride* culture in step (3) includes: activating and subculturing *Trichoderma viride* (strain number: GDMCC NO.3.443), inoculating it into a PDA slant culture medium, expanding the culture to obtain a seed culture; adding sterile water to dissolve the *Trichoderma viride* spores in the seed culture into the sterile water, obtaining a *Trichoderma viride* spore concentration of 10. 7 ~10 8 CFU / mL Trichoderma viride spore suspension; add 1 mL of Trichoderma viride spore suspension to 300 mL of liquid culture medium, and culture at 26-30℃ with shaking at 180-200 r / min for 72-80 h to obtain Trichoderma viride solution.

[0017] In some embodiments, the liquid culture medium is formulated as follows: 100 mL sterile water, 2–2.5 g glucose, 2–2.5 g peptone, 0.6–0.7 g potassium dihydrogen phosphate, 0.2–0.3 g magnesium sulfate, 0.5–0.6 g ammonium sulfate, and 0.1–0.2 g calcium chloride.

[0018] In some embodiments, in step (3), the rotation speed of the oscillation culture is 180-200 r / min.

[0019] In some embodiments, step (4) specifically involves: sterilizing the fermentation broth; then adding an equal amount of deionized water from step (2), magnetically stirring to disperse evenly, adjusting the pH to 7, adding 2.5% of the bran weight of high-temperature α-amylase, and performing enzymatic hydrolysis at 95°C for 3.5 hours; after the enzymatic hydrolysis is completed, adjusting the pH to 7, adding 2% of the bran weight of neutral protease for 2 hours of enzymatic hydrolysis; after the enzymatic hydrolysis is completed, adjusting the pH to 11, and performing alkaline hydrolysis at 40°C for 1.5 hours.

[0020] In some embodiments, the method for extracting water-soluble dietary fiber from the filtrate in step (6) includes the following steps: adjusting the pH of the filtrate to 4.5 with 1 mol / L HCl aqueous solution, letting it stand for 1 h, centrifuging at 4000 r / min for 10 min, adjusting the pH of the supernatant to 7 with 1 mol / L NaOH aqueous solution, rotary evaporating under reduced pressure, adding four times the volume of anhydrous ethanol overnight, centrifuging at 4000 r / min for 10 min, discarding the supernatant, and freeze-drying to obtain water-soluble dietary fiber.

[0021] In some embodiments, in step (6), water-insoluble dietary fiber is extracted from the precipitate, including the following steps: the precipitate obtained in step (5) is washed with water until the pH reaches 7, centrifuged, and freeze-dried to obtain water-insoluble dietary fiber.

[0022] On the other hand, it provides high-quality water-soluble and water-insoluble dietary fiber obtained by the aforementioned process.

[0023] Beneficial effects:

[0024] (1) The method of the present invention uses a combination of processes, including pre-crushing, ultra-fine crushing followed by airflow sieving, green mold fermentation, high-temperature α-amylase and neutral protease hydrolysis, alkaline hydrolysis, and hydrogen peroxide decolorization, to extract dietary fiber from wheat bran. By combining specific green mold fermentation parameters and hydrogen peroxide decolorization parameters, high-quality dietary fiber was successfully obtained: the purity of soluble dietary fiber reached 37.1%, and the purity of insoluble dietary fiber reached 67.3%. Compared with the traditional enzymatic hydrolysis process, the purity of soluble dietary fiber increased by 3.2 times, the purity of insoluble dietary fiber increased by 54.7%, the dietary fiber color was lighter, the whiteness increased by 30-40%, the sensory quality was better, the use value was increased, and the functionality of dietary fiber was better—DPPH and FRAP antioxidant activity increased by 1-2 times, enzyme inhibition increased by 35%-45%, nitrite adsorption capacity increased by 1.4-1.78 times, and cholesterol adsorption capacity increased by 1-3 times. The improvement of the above properties is the result of the synergistic effect of multiple factors, and the degree of improvement is far beyond the expectations of those skilled in the art.

[0025] (2) This invention found that, based on the method of this invention, different decolorization processes have a significant impact on the decolorization effect and the purity of insoluble and soluble dietary fiber: after decolorization with hydrogen peroxide, the L value of soluble dietary fiber increased to 90.85±1.82, and the L value of insoluble dietary fiber increased to 75.69±1.28. Compared with other decolorization processes, the color is whiter, and the purity loss of insoluble and soluble dietary fiber is lower than that of Comparative Example 1 obtained without decolorization treatment; by combining hydrogen peroxide decolorization process with green wood enzyme fermentation process, the purity loss of insoluble and soluble dietary fiber is lower. It exhibits high purity of insoluble and soluble dietary fiber and excellent whitening and decolorizing properties: the L-value of insoluble dietary fiber is further improved. Compared with the traditional extraction process of insoluble and soluble dietary fiber, the purity of soluble dietary fiber increased from 11.5% to 37.1%; the L-value of soluble dietary fiber increased from 65.86±1.23 to 91.61±3.31; the purity of insoluble dietary fiber increased from 43.5% to 67.3%, and the L-value of insoluble dietary fiber increased from 56.66±1.04 to 88.29±0.9.

[0026] (3) The *Trichoderma viride* used in this invention has the advantages of rapid growth, short enzyme production cycle, and high cellulase activity. The cellulase mainly consists of exo-β-1,4-glucan-biose hydrolase and endo-β-1,4-glucan-biose hydrolase. These enzymes degrade cellulose into smaller molecules, forming molecules with varying degrees of polymerization, which can effectively increase the content of soluble dietary fiber. This invention uses wheat bran as raw material, inoculates it with *Trichoderma viride*, and extracts dietary fiber through fermentation, thereby increasing the content of soluble dietary fiber. Simultaneously, its metabolites have high safety. This invention employs a very simple culture medium formula and a single-strain fermentation process, making the preparation method simpler, safer, and more feasible, providing an effective way for the comprehensive utilization of wheat bran.

[0027] (4) The present invention uses an ultra-fine grinding method to process wheat bran. It breaks the internal cohesion of the material through physical mechanical shearing and extrusion, and quickly grinds the material into ultra-fine powder with uniform particles, thereby minimizing the loss and destruction of organic nutrients in the material and maximizing the utilization of raw materials.

[0028] (5) The process employed in this invention is simple and convenient, and the fermentation method is safe and feasible. The raw materials used in this invention are byproducts of wheat production and processing. Through rational utilization, their value is increased. At the same time, the equipment used in this invention is mature, the method is simple, the production cycle is short, and it is easy to realize industrial production, and it meets people's general requirements for natural, safe, and healthy food. This invention can simultaneously prepare soluble and insoluble dietary fiber, making the most comprehensive utilization of the raw materials. Attached Figure Description

[0029] Figure 1This is a process flow diagram in some embodiments of the present invention;

[0030] Figure 2 Photographs of the insoluble and soluble dietary fiber obtained in Example 1, Comparative Example 2, and Comparative Example 1. Detailed Implementation

[0031] Raw materials and reagents used in the embodiments and comparative examples of this invention:

[0032] Wheat bran was purchased from Qinghai Xindingxiang Grain and Oil Co., Ltd.

[0033] Trichoderma viride Persoon, purchased from Guangdong Provincial Microbial Culture Collection Center, strain number: GDMCC NO.3.443;

[0034] Thermoresistant α-amylase, 20,000 u / g, purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0035] Neutral protease, 50,000 u / g, purchased from Beijing Solarbio Technology Co., Ltd.

[0036] Potato glucose agar (PDA) slant culture medium, 30% hydrogen peroxide solution, sodium hydroxide, hydrochloric acid, sulfuric acid, potassium dihydrogen phosphate, magnesium sulfate, ammonium sulfate, calcium chloride, etc., were purchased from Sinopharm Chemical Reagent Co., Ltd. All the above chemical reagents were of analytical grade.

[0037] The liquid culture medium is formulated as follows: 100 mL sterile water, 2 g glucose, 2 g peptone, 0.65 g potassium dihydrogen phosphate, 0.25 g magnesium sulfate, 0.6 g ammonium sulfate, and 0.15 g calcium chloride.

[0038] Instruments and equipment used in the embodiments and comparative examples of this invention:

[0039] AX224ZH / E Electronic Analytical Balance, Ohaus Instruments (Changzhou) Co., Ltd.

[0040] UltraScan Pro1166 high-precision spectrophotometer, Hunterlab, USA;

[0041] BSC-250 constant temperature and humidity chamber, Shanghai Boxun Medical Bio-Instrument Co., Ltd.

[0042] IKA RV10 digital display vertical rotary evaporator, IKA Instruments GmbH, Germany;

[0043] Christ explosion-proof freeze dryer, from the German company Christ.

[0044] UV-3200 ultraviolet spectrophotometer, Shanghai Meipuda Instrument Co., Ltd.;

[0045] SH-100 microplate reader, HITACHI Corporation, Japan;

[0046] Kjeldahl nitrogen analyzer, Jinan Haineng Instrument Co., Ltd.

[0047] Example 1

[0048] (1) Pretreatment of wheat bran: Wheat bran is pre-crushed, ultra-finely pulverized (120 mesh), and sieved through an air sieve (120 mesh) to obtain wheat bran powder;

[0049] (2) Preparation of wheat bran liquid culture medium: Mix wheat bran powder, potassium dihydrogen phosphate, magnesium sulfate, ammonium sulfate and calcium chloride, add deionized water at a solid-liquid ratio of 1:10 (w / v, g / mL), mix well, adjust pH to 3.5, sterilize (121℃, 0.1MPa, 15min) to obtain wheat bran liquid culture medium; In the wheat bran liquid culture medium: the amount of wheat bran powder added is 10g / 100g water, the amount of potassium dihydrogen phosphate added is 0.65g / 100g water, the amount of magnesium sulfate added is 0.25g / 100g water, the amount of ammonium sulfate added is 0.6g / 100g water and the amount of calcium chloride added is 0.15g / 100g water;

[0050] (3) Preparation of *Trichoderma viride* culture: *Trichoderma viride* (strain number: GDMCC NO.3.443) was activated, subcultured, and inoculated into PDA slant medium. The culture was expanded at 28℃ for one week to obtain the inoculum. 2 mL of sterile water was added to the PDA slant medium to dissolve the *Trichoderma viride* spores, resulting in a spore concentration of 10⁻⁶. 8 CFU / mL Trichoderma viride spore suspension; 1 mL of Trichoderma viride spore suspension was added to 300 mL of liquid culture medium and cultured at 28 °C with shaking at 200 r / min for 72 h to obtain Trichoderma viride solution;

[0051] (4) Inoculation and culture: Inoculate the green Trichoderma suspension into the wheat bran liquid culture medium at an inoculation amount of 10% (volume ratio), shake well and place in a constant temperature incubator for culture. The fermentation broth culture conditions are 28℃, 72h, and 200r / min.

[0052] (5) Sterilize the fermentation broth (121℃, 0.1MPa, 15min); then add an equal amount of deionized water from step (2), stir magnetically to disperse evenly, adjust the pH to 7 with 1mol / L HCl aqueous solution or NaOH aqueous solution, add 2.5% (wheat bran mass) high-temperature α-amylase, and perform enzymatic hydrolysis at 95℃ for 3.5h; after enzymatic hydrolysis, adjust the pH to 7 with 1mol / L HCl aqueous solution or NaOH aqueous solution, add 2% (wheat bran mass) neutral protease for 2h enzymatic hydrolysis; after enzymatic hydrolysis, adjust the pH to 11 with 1mol / L HCl aqueous solution or NaOH aqueous solution, and perform alkaline hydrolysis at 40℃ for 1.5h; add 30% hydrogen peroxide solution with a volume ratio of 2.5%, decolorize in a 35℃ water bath for 2.5h, filter, and obtain filtrate and precipitate;

[0053] (6) Take the filtrate obtained in step (5) and adjust the pH to 4.5 with 1 mol / L HCl aqueous solution. Let it stand for 1 h, centrifuge at 4000 r / min for 10 min, take the supernatant and adjust the pH to 7 with 1 mol / L NaOH aqueous solution. After rotary evaporation under reduced pressure, add four times the volume of anhydrous ethanol and let it stand overnight. Centrifuge at 4000 r / min for 10 min, discard the supernatant, freeze-dry (first pre-freeze at -20℃ for 24 h, then freeze at -80℃ for 2 h, and then freeze-dry for 96 h) to obtain water-soluble dietary fiber (soluble dietary fiber);

[0054] (7) Take the precipitate obtained in step (5) and wash it until the pH reaches 7. After centrifugation and freeze drying (pre-freeze at -20℃ for 24 hours, freeze at -80℃ for 2 hours, and then freeze dry for 96 hours), water-insoluble dietary fiber (insoluble dietary fiber) is obtained.

[0055] Comparative Example 1

[0056] Compared to Example 1, the ultrafine grinding, *Trichoderma viride* fermentation, and decolorization were not performed. The specific steps are as follows:

[0057] (1) Take wheat bran, add deionized water at a solid-liquid ratio of 1:20 (w / v, g / mL), stir magnetically to disperse evenly, adjust the pH to 7 with 1 mol / L HCl aqueous solution or NaOH aqueous solution, add 2% (wheat bran mass) high-temperature α-amylase, and carry out enzymatic hydrolysis at 95℃ for 4 h. After the enzymatic hydrolysis, adjust the pH to 7 with 1 mol / L HCl aqueous solution or NaOH aqueous solution, add 1.5% (wheat bran mass) neutral protease to enzymatic hydrolysis for 2 h, adjust the pH to 11 with 1 mol / L NaOH aqueous solution, and carry out alkaline hydrolysis at 40℃ for 1.5 h. Filter to obtain filtrate and precipitate;

[0058] (2) Take the filtrate obtained in step (1) and adjust the pH to 4.5 with 1 mol / L HCl aqueous solution. Centrifuge at 4000 r / min for 10 min. Take the supernatant and adjust the pH to 7 with 1 mol / L NaOH aqueous solution. After rotary evaporation under reduced pressure, add four times the volume of anhydrous ethanol and let it sit overnight. Centrifuge at 4000 r / min for 10 min. Discard the supernatant and freeze dry to obtain water-soluble dietary fiber.

[0059] (3) Take the precipitate obtained in step (1), wash it with water until the pH reaches 7, centrifuge and freeze dry to obtain water-insoluble dietary fiber.

[0060] Comparative Example 2: Screening and Process Optimization of Decolorizing Agents

[0061] Compared with Comparative Example 1, a decolorization process was added, and the specific steps are as follows:

[0062] (1) Take wheat bran, add deionized water at a solid-liquid ratio of 1:20 (w / v, g / mL), stir magnetically to disperse evenly, adjust the pH to 7 with 1 mol / L HCl aqueous solution or NaOH aqueous solution, add 2% (wheat bran mass) high-temperature α-amylase, and carry out enzymatic hydrolysis at 95℃ for 4 h. After the enzymatic hydrolysis, adjust the pH to 7 with 1 mol / L HCl aqueous solution or NaOH aqueous solution, add 1.5% (wheat bran mass) neutral protease for 2 h, and after the enzymatic hydrolysis, adjust the pH to 11 with 1 mol / L NaOH aqueous solution, and carry out alkaline hydrolysis at 40℃ for 1.5 h.

[0063] (2) Add 2.5% of 30% hydrogen peroxide solution by volume, decolorize in a water bath at 35°C for 2.5 hours, filter, and obtain filtrate and precipitate;

[0064] (3) Take the filtrate obtained in step (2) and adjust the pH to 4.5 with 1 mol / L HCl aqueous solution. Centrifuge at 4000 r / min for 10 min. Take the supernatant and adjust the pH to 7 with 1 mol / L NaOH aqueous solution. After rotary evaporation under reduced pressure, add four times the volume of anhydrous ethanol and let it sit overnight. Centrifuge at 4000 r / min for 10 min. Discard the supernatant and freeze dry to obtain water-soluble dietary fiber.

[0065] (4) Take the precipitate obtained in step (2), wash it with water until the pH reaches 7, centrifuge and freeze dry to obtain water-insoluble dietary fiber.

[0066] Comparative Example 2-1

[0067] Compared with Comparative Example 2, the phrase "add 2.5% of 30% hydrogen peroxide solution by volume" was replaced with "add 2.5% sodium hypochlorite solution by volume".

[0068] Comparative Example 2-2

[0069] Compared with Comparative Example 2, the phrase "add 2.5% of 30% hydrogen peroxide solution by volume, decolorize in a water bath at 35°C for 2.5 h, filter, and obtain filtrate and precipitate" was replaced with "filter, obtain filtrate and precipitate, add 2.5% (w / v, g / mL) activated carbon to the filtrate, allow to stand for 24 h for adsorption, centrifuge, and take the filtrate".

[0070] Table 1. Dietary fiber from different decolorization methods

[0071]

[0072] The color of dietary fiber affects its sensory quality and usability; therefore, a decolorization process is performed. The colors before and after the decolorization process are shown in Table 1. Figure 2 As shown.

[0073] The results showed that, compared with Comparative Example 2, Comparative Example 2-1, and Comparative Example 2-2, different decolorization processes had a significant impact on the decolorization effect and the purity of insoluble and soluble dietary fiber: the decolorization effect of activated carbon adsorption in Comparative Example 2-2 was not obvious; after sodium hypochlorite decolorization in Comparative Example 2-1, the L-value of soluble dietary fiber increased from 65.86±1.23 to 80.59±0.44, and the L-value of insoluble dietary fiber increased from 56.66±1.04 to 70.59±0.59, which was worse than the effect of hydrogen peroxide decolorization in Comparative Example 2, and the purity loss of the obtained insoluble and soluble dietary fiber was high. It is noteworthy that after decolorization with hydrogen peroxide, the L-value of soluble dietary fiber increased to 90.85±1.82, and the L-value of insoluble dietary fiber increased to 75.69±1.28. Compared with other decolorization processes, the color is whiter and the purity loss of insoluble and soluble dietary fiber is lower than that of Comparative Example 1 obtained without decolorization. Comparing Example 1 and Comparative Example 2, it can be found that Example 1, by combining hydrogen peroxide decolorization and green wood enzyme fermentation, simultaneously achieved high purity of insoluble and soluble dietary fiber and excellent whitening and decolorizing performance: the L value of insoluble dietary fiber was further improved, increasing from 75.69±1.28 in Comparative Example 2 to 88.29±0.9; compared with the traditional extraction process of insoluble and soluble dietary fiber in Comparative Example 1, the purity of soluble dietary fiber increased from 11.5% to 37.1%; the L value of soluble dietary fiber increased from 65.86±1.23 to 91.61±3.31; the purity of insoluble dietary fiber increased from 43.5% to 67.3%, and the L value of insoluble dietary fiber increased from 56.66±1.04 to 88.29±0.9.

[0074] Based on preliminary experiments and single-factor experiments, the initial decolorization conditions were determined to be: hydrogen peroxide addition of 2.5% of the total volume, decolorization time of 2 hours, and decolorization temperature of 40℃. An orthogonal experimental design (L9(3,4)) was used to investigate the three factors: hydrogen peroxide addition, decolorization time, and decolorization temperature. Each factor had three levels: hydrogen peroxide addition of 2, 2.5, and 3; decolorization times of 1.5 hours, 2 hours, and 2.5 hours; and decolorization temperatures of 35℃, 40℃, and 45℃. The decolorization results were evaluated using the color difference of soluble dietary fiber as the evaluation index. The results are shown in Table 2.

[0075] Table 2 Results of orthogonal experiments on decolorization process

[0076]

[0077] Table 2 shows that the order of influence of factors on the L value is C>A>B, i.e., decolorization temperature>amount added>decolorization time. The optimal scheme is A2B3C1, i.e., hydrogen peroxide addition amount is 2.5%, time is 2.5h, and temperature is 35℃. Since the effect of decolorization time is relatively low, and decolorization will reduce the purity of dietary fiber to a certain extent (as shown in Table 1), the decolorization time can be appropriately shortened according to the actual situation.

[0078] Comparative Example 3: Selection and Optimization of the Fermentation Process for Trichoderma viride

[0079] Compared with Comparative Example 2, a green Trichoderma fermentation process was added. The specific steps are shown in steps (2) to (6) of Example 1, where wheat bran powder was replaced with wheat bran.

[0080] Based on preliminary experiments and single-factor experiments, the initial fermentation conditions were determined to be: fermentation pH 4.5, inoculum size 10%, and fermentation time 72 hours. An orthogonal experimental design (L9(3,4)) was used to investigate the fermentation pH, Trichoderma inoculum size, and fermentation time. Each factor had three levels: fermentation pH 3.5, 4.5, and 5.5; Trichoderma inoculum size 7%, 10%, and 13%; and fermentation time 48 h, 60 h, and 72 h. The effects of different factors on the fermentation results of *Trichoderma viride* were evaluated using soluble dietary fiber content and total dietary fiber as comprehensive indicators. The results are shown in Table 3.

[0081] Table 3. Orthogonal Experiment and Results Analysis for Optimization of *Trichoderma viride* Process

[0082]

[0083] Note: Overall score = (Soluble dietary fiber content / MAX (soluble dietary fiber content)) × 80 + (Insoluble dietary fiber content / MAX (insoluble dietary fiber content)) × 20

[0084] Table 3 shows that the order of influence of the three factors on the overall score is A>B>C. The optimal conditions for *Trichoderma viride* are A3B1C3, namely, an inoculum size of 13%, a fermentation broth pH of 3.5, and a fermentation time of 72 hours. Since the change in inoculum size has little effect on the experimental results, and adhering to the principle of cost-saving, the final fermentation conditions were determined to be an inoculum size of 10%, a fermentation broth pH of 3.5, and a fermentation time of 72 hours.

[0085] Comparative Example 4: Ultrafine Grinding Process

[0086] Compared with Comparative Example 2, an ultrafine pulverization process was added, and the specific steps are as follows:

[0087] (1) Pretreatment of wheat bran: Wheat bran is pre-crushed, ultra-finely pulverized (120 mesh), and sieved through an air sieve (120 mesh) to obtain wheat bran powder;

[0088] The remaining steps are the same as in Comparative Example 2.

[0089] Test case

[0090] The following indicators were measured for the freeze-dried dietary fiber in some of the above-mentioned examples and comparative examples.

[0091] 1. Comparison of dietary fiber purity after different processing techniques

[0092] The purity of dietary fiber after different processing methods is shown in Table 4.

[0093] Comparative Example 1 shows dietary fiber prepared using a conventional enzymatic hydrolysis process, with a soluble dietary fiber purity of 11.5% and an insoluble dietary fiber purity of 43.5%. Comparative Example 2 shows dietary fiber treated with an additional decolorization process; the results indicate that decolorization reduces the purity of soluble dietary fiber. Comparative Example 3 further incorporates a *Trichoderma viride* fermentation process; after fermentation, the soluble dietary fiber purity is 21.8%, and the insoluble dietary fiber purity is 47.4%. Comparative Example 3 also incorporates an ultrafine grinding process; after ultrafine grinding and enzymatic hydrolysis, the soluble dietary fiber purity is... The purity of soluble dietary fiber was 15.8%, and the purity of insoluble dietary fiber was 49.2%. Example 1 describes the combined ultrafine grinding and *Trichoderma viride* fermentation process of this invention. The results showed that the purity of soluble dietary fiber was 37.1%, and the purity of insoluble dietary fiber was 67.3%. Compared with wheat bran raw material, the purity of soluble dietary fiber increased by 19 times, and the purity of insoluble dietary fiber increased by 58.7%. Compared with ordinary enzymatic hydrolysis process, the purity of soluble dietary fiber increased by 3.2 times, and the purity of insoluble dietary fiber increased by 54.7%.

[0094] Table 4. Purity of soluble dietary fiber before and after different processing techniques

[0095]

[0096] 2. Functional characteristic determination

[0097] The dietary fiber prepared in Example 1 and Comparative Example 1 of this invention was functionally evaluated, including water-holding capacity, swelling capacity, cholesterol adsorption capacity, and sodium cholate adsorption capacity, etc., by means of the following methods:

[0098] ① Water retention

[0099] Accurately weigh 1g of dietary fiber sample and place it in a 50mL centrifuge tube, recording the total mass as m1. Add 20mL of deionized water, shake at room temperature for 30min, centrifuge at 4000rpm for 10min, discard the supernatant, and weigh the centrifuge tube and precipitate, recording the mass as m2. Perform three parallel tests for each sample and take the average value. Calculate the water-holding capacity according to formula (3-6):

[0100]

[0101] ②Expansion capacity

[0102] The sample (0.5 g) was soaked in 6.0 mL of distilled water in a 10 mL graduated cylinder. The bran was soaked in the graduated cylinder for 24 hours, during which time the bran absorbed water and swelled. The volume occupied by the swollen bran was then recorded. The analysis was performed three times in duplicate.

[0103] Water-holding capacity and swelling capacity are important functional properties of dietary fiber. Good water-holding capacity can prevent food from dehydrating and shrinking, and it also has a laxative effect in the intestines. Experiments show that Example 1 exhibits better physicochemical properties than Comparative Example 1. As can be seen from Table 5, the water-holding capacity and swelling capacity of dietary fiber in Example 1 are significantly increased, with water-holding capacity being 1.78 times that of Comparative Example 1 and swelling capacity being 1.64 times that of Comparative Example 1.

[0104] Table 5. Water-holding capacity and swelling capacity of insoluble dietary fiber

[0105]

[0106] ③ Determination of polyphenol and flavonoid content

[0107] Wheat bran powder (0.5 g) was extracted four times with 10 mL of 80% methanol at room temperature for 30 min each time. After centrifugation at 4000 rpm for 10 min, the supernatants were mixed and brought to a final volume of 50 mL with 80% methanol.

[0108] Polyphenol content determination: Take 125 μL of sample extract, add 500 μL of distilled water and 125 μL of Folin-Ciocalteu, shake well, react at room temperature for 6 min, add 1.25 mL of 7% Na2CO3 solution, then add 1 mL of distilled water, and place at room temperature in the dark for 1.5 h. Measure the absorbance at a wavelength of 765 nm. At the same time, prepare a standard curve using gallic acid. The content is expressed as mg GA / 100g DW.

[0109] Determination of flavonoids: Take 100 μL of sample extract, add 40 μL of 0.1 mol / L aluminum trichloride solution, shake well, then add 60 μL of 1 mol / L potassium acetate solution, shake well, and let stand at room temperature in the dark for 30 min. Measure the absorbance at a wavelength of 420 nm. At the same time, prepare a standard curve using rutin. The results are expressed as mg TE / 100g DW.

[0110] ④ Antioxidant performance determination

[0111] DPPH free radical scavenging ability

[0112] 100 μL of polyphenol solution was pipetted into a 96-well plate, and 100 μL of 4 mg / mL DPPH solution was added. Anhydrous ethanol was used instead of DPPH working solution and mixed with the sample as a blank group. Anhydrous ethanol and DPPH working solution were mixed as a control group. After reacting in the dark for 30 min, the absorbance was measured at a wavelength of 517 nm, and the free radical scavenging capacity of DPPH was calculated according to the formula.

[0113]

[0114] ABTS free radical scavenging ability

[0115] Pipette 30 μL of sample solution into a 96-well plate, add 270 μL of ABTS working solution (8 mmol / L ABTS solution and 19.6 mmol / L potassium persulfate solution are mixed in a 7:1 volume ratio, allowed to stand in the dark at room temperature for 12–16 h, and then diluted with deionized water or ethanol until the absorbance at 734 nm reaches 0.70 ± 0.02, which is the ABTS working solution), react in the dark for 6 min, and then measure the absorbance at a wavelength of 734 nm. Calculate the ABTS free radical scavenging capacity according to the formula.

[0116]

[0117] Evaluation of Ferric Reduction Ability (FRAP)

[0118] A 36 μL sample was pipetted into a 96-well plate, and 270 μL of FRAP working solution was added (each 1 L of FRAP working solution contained 3.1 g anhydrous sodium acetate, 16 mL acetic acid, 0.31 mg dithiothreitol, 2 mL 2 mol / L HCl, and 0.54 g FeCl3·6H2O). The plate was incubated at 37 °C in the dark for 8 min, and the absorbance was measured at 593 nm. Simultaneously, a standard curve was plotted using FeSO4 standard solution instead of the sample.

[0119] The polyphenol and flavonoid contents of dietary fiber in different embodiments and comparative examples are shown in Table 6. In Example 1, the polyphenol content of soluble dietary fiber increased and the flavonoid content decreased; the polyphenol and flavonoid contents of insoluble dietary fiber both increased. Compared with Comparative Example 1, the antioxidant properties of dietary fiber in Example 1 showed different degrees of increase in DPPH, ABTS, and FRAP. Among them, the antioxidant properties of ABTS did not change significantly, soluble dietary fiber showed high DPPH antioxidant properties, and insoluble dietary fiber had high FRAP antioxidant properties.

[0120] Table 6. Polyphenol and flavonoid content and antioxidant properties of dietary fiber

[0121]

[0122] ⑤ Glucose absorption capacity

[0123] Weigh 0.5g of dietary fiber sample, add 100mL of 100mmol / L glucose solution, stir magnetically at 37℃ for 5h, centrifuge at 4000r / min for 20min, with glucose solution as blank, take the supernatant and determine the glucose content using the GOPOD method, and calculate the glucose adsorption capacity.

[0124]

[0125] In the formula: C0 is the glucose concentration in the blank, mmol / L; C1 is the glucose concentration in the sample, mmol / L; V is the volume of glucose solution added; M is the sample mass.

[0126] ⑥ In vitro adsorption capacity of sodium cholate

[0127] In vitro adsorption of sodium cholate by bran dietary fiber: 100 mL of 0.15 mol / L NaCl solution containing 0.2 g sodium cholate was added to a 250 mL Erlenmeyer flask, the pH was adjusted to 7, 1.0 g of dietary fiber was added, and the mixture was stirred until fully dissolved. The mixture was then shaken in a 37°C water bath for 3 h. After shaking, 0.5 mL of the reaction solution was transferred to a 25 mL stoppered test tube, and 0.5 mL of 45% sulfuric acid solution and 0.3% furfural solution were added. The tube was then placed in a 37°C water bath for 30 min. After cooling, the absorbance was measured at 620 nm using distilled water as a reference. A standard curve was simultaneously established using sodium cholate.

[0128] ⑦ Assay for pancreatic lipase activity inhibition

[0129] Weigh 0.5 g of dietary fiber and add it to 10 mL of olive oil, 50 mL of sodium phosphate buffer (0.1 M, pH 7.2), and 10 mL of pancreatic lipase solution. Mix well. Prepare a pancreatic lipase solution by adding 7.1 mL of pancreatic lipase to the 10 mL sodium phosphate buffer. After incubating in a 37°C water bath for 1 h, place the test tube in a boiling water bath for 10 min to stop the reaction. Finally, titrate with 0.1 mol / L sodium hydroxide to determine the amount of free fatty acids released. The inhibitory activity (%) on lipase is defined as the percentage reduction in the rate of free fatty acid production relative to the control group (no fiber, 39.7 mg oleic acid / hour).

[0130]

[0131] Note: V is the volume of NaOH consumed without adding the sample, in mL; V1 is the volume of NaOH consumed with the addition of dietary fiber, in mL.

[0132] ⑧ α-Amylase Activity Inhibition Assay

[0133] Weigh 0.5g of dietary fiber, add 20mL of 4% potato starch solution, then add 300μL of α-amylase, mix and hydrolyze at 37℃ for 1h, inactivate the enzyme at 100℃ for 10min, centrifuge at 4000r / min for 20min, take the supernatant and determine the glucose content using the GOPOD method, and calculate the α-amylase activity inhibition.

[0134]

[0135] In the formula: λ0 is the glucose concentration in the blank, mmol / L; λ1 is the glucose concentration in the sample, mmol / L.

[0136] Table 7 shows the glucose and nitrite adsorption capacities of dietary fiber from different examples and comparative examples. Compared with Comparative Example 1, Example 1 exhibited higher adsorption capacity and enzyme inhibition. Specifically, the glucose adsorption capacity of soluble and insoluble dietary fiber in Example 1 increased by 1.31 and 1.5 times, respectively, and the sodium cholate adsorption capacity increased by 1.52 and 1.31 times, respectively. The enzyme inhibition capacities of soluble and insoluble dietary fiber differed slightly. Insoluble dietary fiber showed more significant inhibition of pancreatic lipase, increasing from 13.5% to 38.2%, while soluble dietary fiber showed higher inhibition of α-amylase, increasing from 11.5% to 37.8%, both approximately three-fold increases.

[0137] Table 7 Adsorption and Enzyme Inhibition Capacity of Dietary Fiber

[0138]

[0139] ⑨ Nitrite adsorption capacity

[0140] Construction of the nitrite ion standard curve: Accurately measure 0.20 mL, 0.40 mL, 0.60 mL, 0.80 mL, 1.00 mL, 1.20 mL, and 1.50 mL of 100 μmol / L NaNO₂ solution into seven 50 mL colorimetric tubes. Then, add 2 mL of 0.4% p-aminosulfonic acid solution to each tube, mix thoroughly, and let stand for 3-5 min. Next, add 1 mL of 0.2% ethylenediamine hydrochloride solution to each tube, then add distilled water to the mark, mix well, and let stand for 15 min. Zero the spectrophotometer using the zero tube and measure the absorbance at 538 nm to construct the standard curve.

[0141] Determination of the adsorption capacity of bran dietary fiber for nitrite ions: 50 mL of 100 μmol / L NaNO2 solution was added to a 50 mL flask, and the pH was adjusted to 2 and 7. After adding 0.5 g of dietary fiber and controlling the temperature at 37 °C, the mixture was magnetically stirred for 2 h, centrifuged at 3500 r / min for 5 min, and the concentration of residual nitrite ions in the solution after adsorption was measured.

[0142] ⑩ Cholesterol binding capacity

[0143] Accurately weigh 0.5000g of cholesterol, dissolve it in deionized water, and transfer it to a 100mL volumetric flask and dilute to volume. Accurately pipette 2.0mL, 3.0mL, 4.0mL, 5.0mL, and 6.0mL of this solution into 10mL volumetric flasks and dilute to volume. Accurately pipette 0.02mL of each of the different concentrations of standard solutions, add 0.38mL of glacial acetic acid, 1.5mL of 0.1mg / mL o-phthalaldehyde-glacial acetic acid solution, and 1mL of concentrated sulfuric acid, respectively. Measure the concentrations using a spectrophotometer at a wavelength of 550nm and plot a standard curve.

[0144] Separate the egg white and yolk from one egg. Accurately weigh the volume of the yolk and add distilled water in a 1:9 ratio, stirring well. Accurately weigh 0.5g of dietary fiber sample into two beakers, add 10mL of the above egg yolk solution to each, mix well, adjust the pH to 2.0 (simulating the stomach environment) and 7.0 (simulating the small intestine environment), and incubate at 37℃ with water bath shaking for 2 hours. Centrifuge at 4000r / min for 20 minutes, and determine the cholesterol content according to the o-phthalaldehyde method.

[0145]

[0146] In the formula: M0 is the cholesterol content before adsorption, mg; C1 is the cholesterol content after adsorption, mg; M is the sample mass.

[0147] Table 8. Adsorption and binding capacity of dietary fiber for nitrite and cholesterol.

[0148]

[0149] Dietary fiber can reduce the absorption and utilization of excess triglycerides, cholesterol, and bile salts in the small intestine, lower blood cholesterol levels, promote cholesterol metabolism, and reduce blood lipid levels, thereby reducing the risk of cardiovascular disease. The results are shown in Table 8. Different pH values ​​exhibited different adsorption and binding capacities. At pH 7, the nitrate adsorption capacity increased by 1.76 and 1.57 times, respectively, while at pH 2, although the nitrate adsorption capacity was higher than at pH 7, the change was not significant compared to the control group. The soluble dietary fiber in Example 1 had a higher cholesterol binding capacity at pH 2, while the insoluble dietary fiber had a higher cholesterol binding capacity at pH 7.

[0150] In summary, the dietary fiber prepared by this invention exhibits varying degrees of improvement in properties such as water retention, glucose adsorption capacity, cholesterol adsorption capacity, and nitrite adsorption capacity. These improvements in functional properties have a more positive impact on human diets and product development.

[0151] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A process for extracting high-quality dietary fiber from wheat bran, characterized in that, Includes the following steps: (1) Pretreatment of wheat bran: Wheat bran is pre-crushed, ultra-fine pulverized, and sieved by airflow sieve to obtain wheat bran powder; the mesh size of ultra-fine pulverization is 120 mesh, and the mesh size of airflow sieve is 120 mesh. (2) Preparation of wheat bran liquid culture medium: Mix wheat bran powder, potassium dihydrogen phosphate, magnesium sulfate, ammonium sulfate and calcium chloride, add deionized water, mix well, adjust pH to 3.5, sterilize to obtain wheat bran liquid culture medium; The components of wheat bran liquid culture medium are 100 g water, 9~10 g wheat bran powder, 0.6~0.7 g potassium dihydrogen phosphate, 0.2~0.3 g magnesium sulfate, 0.5~0.6 g ammonium sulfate and 0.1~0.2 g calcium chloride; (3) Inoculate the *Trichoderma viride* solution into the wheat bran liquid culture medium at an inoculation rate of 10% by volume, shake well, and place in a constant temperature incubator at 26-30℃ for 72 h to obtain the fermentation broth; wherein, the preparation method of the *Trichoderma viride* solution includes: dissolving *Trichoderma viride* spores in sterile water, wherein the *Trichoderma viride* strain number is GDMCC NO.3.443, and the *Trichoderma viride* spore concentration is 10 7 ~10 8 A CFU / mL suspension of *Trichoderma viride* spores was prepared; 1 mL of the *Trichoderma viride* spore suspension was added to 300 mL of liquid culture medium and cultured at 26-30℃ with shaking at 180-200 rpm for 72-80 h to obtain *Trichoderma viride* culture; the liquid culture medium consisted of 100 mL sterile water, 2-2.5 g glucose, 2-2.5 g peptone, 0.6-0.7 g potassium dihydrogen phosphate, 0.2-0.3 g magnesium sulfate, 0.5-0.6 g ammonium sulfate, and 0.1-0.2 g calcium chloride. (4) Sterilize the fermentation broth; then, enzymatically hydrolyze it with high-temperature α-amylase and neutral protease in sequence; then, perform alkaline hydrolysis; the enzyme activity of high-temperature α-amylase is 20,000 U / g, and the enzyme activity of neutral protease is 50,000 U / g; Specifically: the fermentation broth was sterilized; then an equal amount of deionized water from step (2) was added, and the mixture was magnetically stirred and dispersed evenly. The pH was adjusted to 7, and 2.5% of the bran weight of high-temperature α-amylase was added. Enzymatic hydrolysis was carried out at 95 ℃ for 3.5 h. After the enzymatic hydrolysis was completed, the pH was adjusted to 7, and 2% of the bran weight of neutral protease was added for 2 h of enzymatic hydrolysis. After the enzymatic hydrolysis was completed, the pH was adjusted to 11, and alkaline hydrolysis was carried out at 40 ℃ for 1.5 h. (5) Add 2.5% by volume of 30% hydrogen peroxide solution, decolorize in a water bath at 35 ℃ for 2.5 h, filter, and obtain filtrate and precipitate; (6) Extract water-soluble dietary fiber from the filtrate; extract water-insoluble dietary fiber from the precipitate.

2. The process according to claim 1, characterized in that, The sterilization conditions for steps (2) and (4) are 121 °C, 0.1 MPa, and 15-20 min.

3. The process according to claim 1, characterized in that, The preparation method of the *Trichoderma viride* culture in step (3) includes: activating *Trichoderma viride*, wherein the strain number of *Trichoderma viride* is GDMCC NO.3.443, and after subculturing, inoculating it into PDA slant medium for expansion culture to obtain a seed culture; adding sterile water to dissolve the *Trichoderma viride* spores in the seed culture into the sterile water to obtain a *Trichoderma viride* spore concentration of 10. 7 ~10 8 CFU / mL Trichoderma viride spore suspension; add 1 mL of Trichoderma viride spore suspension to 300 mL of liquid culture medium, and culture at 26-30℃ with shaking at 180-200 r / min for 72-80 h to obtain Trichoderma viride solution.

4. The process according to any one of claims 1 or 3, characterized in that, In step (3), the rotation speed of the shaking culture is 180~200 r / min.

5. The process according to claim 1, characterized in that, In step (6), the method for extracting water-soluble dietary fiber from the filtrate includes the following steps: the pH of the filtrate is adjusted to 4.5 with 1 mol / L HCl aqueous solution, allowed to stand for 1 h, centrifuged at 4000 r / min for 10 min, the supernatant is adjusted to pH 7 with 1 mol / L NaOH aqueous solution, rotary evaporated under reduced pressure, four times the volume of anhydrous ethanol is added and left overnight, centrifuged at 4000 r / min for 10 min, the supernatant is discarded, and the water-soluble dietary fiber is obtained after freeze-drying.

6. The process according to claim 1, characterized in that, In step (6), water-insoluble dietary fiber is extracted from the precipitate, including the following steps: the precipitate obtained in step (5) is washed with water until the pH reaches 7, centrifuged, and freeze-dried to obtain water-insoluble dietary fiber.

7. High-quality water-soluble dietary fiber and water-insoluble dietary fiber obtained by the process described in any one of claims 1 to 6.

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