A corn bran soluble dietary fiber and a preparation method and application thereof

Abstract

The application provides corn bran soluble dietary fiber and a preparation method and application thereof, and belongs to the technical field of soluble dietary fiber preparation. The application provides a trichoderma reesei strain TE-36 and a method for preparing corn bran soluble dietary fiber by using the strain, and the method comprises the following steps: inoculating seed liquid of the trichoderma reesei strain TE-36 after mixing corn bran powder and a culture medium to carry out fermentation; then, enzyme hydrolysis and alcohol precipitation are carried out to obtain corn bran soluble dietary fiber. The trichoderma reesei strain TE-36 has complete cellulase composition, fast growth speed, high enzyme yield, and can secrete a plurality of high-activity cell wall hydrolytic enzymes, so that the cellulose and hemicellulose macromolecules in the corn bran can be effectively degraded. The preparation method provided by the application is organic fusion of a plurality of treatment modes through a synergistic fermentation technology of bacteria and enzymes, so that the yield, purity and blood sugar reduction function characteristics of the soluble dietary fiber are increased, and the method is suitable for industrialization and popularization.

Description

Technical Field

[0001] This invention belongs to the field of soluble dietary fiber preparation technology, specifically relating to a corn husk soluble dietary fiber, its preparation method, and its application. Background Technology

[0002] Dietary fiber (DF), a carbohydrate analogue, is known as the "seventh nutrient" and is a highly promising food and pharmaceutical ingredient. Numerous studies have confirmed that DF can improve the digestion and absorption of nutrients in the gastrointestinal tract, and treat and intervene in various gastrointestinal diseases. Based on their solubility in water, DF can be broadly classified into soluble dietary fiber (SDF) and insoluble dietary fiber (IDF), with SDF receiving more attention due to its superior physicochemical properties and bioactivity. However, most dietary fiber is insoluble (accounting for approximately 90% of total DF), with a small portion being soluble (less than 10% of total DF).

[0003] Corn is considered one of the most important and highest-yielding grains, possessing significant economic and social value. Corn husks, a major byproduct of corn processing, have not received sufficient attention due to their low water solubility and unpleasant taste, and are often used as feed ingredients. Studies have found that the main components of corn husks are cellulose, hemicellulose, and lignin, with a fiber content of approximately 70%, making it a good source of dietary fiber (DF). However, its SDF content is generally low, and increasing the SDF content in corn husks has been a pressing technical problem in this field. Current technologies primarily focus on the preparation processes of SDF from different sources. In actual production, the generally low SDF content limits the promotion of dietary fiber products to some extent. Existing methods for preparing soluble dietary fiber suffer from problems such as limited variety, low product yield, poor purity, and unsatisfactory functionality. Summary of the Invention

[0004] In view of this, one of the objectives of the present invention is to provide a Trichoderma reesei TE-36 with a complete cellulase system, fast growth rate, high enzyme production, mild growth conditions, and suitability for industrial application. It can secrete a variety of highly active cell wall hydrolases, such as cellulase and xylanase, which can effectively degrade cellulose and hemicellulose macromolecules in corn husks. When used in the preparation of soluble dietary fiber from corn husks, it can effectively improve the yield, purity, and functional activity of soluble dietary fiber from corn husks.

[0005] The second objective of this invention is to provide a method for preparing soluble dietary fiber from corn husks, thereby solving the problems of limited methods, low product yield, and poor purity in current soluble dietary fiber preparation methods.

[0006] A third objective of this invention is to provide the application of the prepared corn husk soluble dietary fiber in the preparation of products that lower blood sugar or help maintain healthy blood sugar levels.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a Trichoderma reesei TE-36, which is classified as Trichoderma reesei and is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No. 41552.

[0009] The present invention also provides the application of the above-mentioned Trichoderma reesei TE-36 in the preparation of soluble dietary fiber from corn husks.

[0010] The present invention also provides a method for preparing soluble dietary fiber from corn husks, comprising the following steps: mixing corn husk powder with a culture medium to obtain a fermentation culture medium; inoculating the above-mentioned Trichoderma reesei TE-36 seed liquid into the fermentation culture medium for fermentation to obtain a fermentation broth; then sequentially performing enzymatic hydrolysis with α-amylase, alkaline protease and glucoamylase, collecting the supernatant, and precipitating with alcohol to obtain soluble dietary fiber from corn husks.

[0011] Preferably, the culture medium comprises 1.0–2.0 g / L K₂HPO₄, 0.3–0.5 g / L MgSO₄·7H₂O, 0.3–0.5 g / L CaCl₂, 0.0015–0.0025 g / L FeSO₄·7H₂O, and 0.0015–0.0025 g / L ZnSO₄·7H₂O; and the concentration of corn husk powder in the fermentation culture medium is 50–66 g / L.

[0012] Preferably, the method for preparing the corn husk powder includes the following steps: ultra-finely pulverizing the corn husk and passing it through a 180-200 mesh sieve, and then irradiating it with γ- to obtain the corn husk powder; the intensity of the γ-irradiation is 8-10 kGy.

[0013] Preferably, the seed culture is obtained by inoculating Trichoderma reesei TE-36 into a seed culture medium and culturing it at 25-28°C for 3-4 days; the seed culture medium comprises 25-30 g / L glucose, 2.5-3.0 g / L NaNO3, 0.8-1.0 g / L K2HPO4, 0.5-1.0 g / L KCl, 0.5-1.0 g / L MgSO4·7H2O, and 0.01-0.03 g / L FeSO4; the volume ratio of the seed culture to the fermentation medium is 8-10:100.

[0014] Preferably, the fermentation is carried out at 28-30℃ for 60-66 hours; or at 28-30℃ for 40-42 hours, followed by the addition of β-glucosidase and continued fermentation for 20-24 hours; the volume ratio of β-glucosidase to fermentation medium is 0.4-0.5:100.

[0015] Preferably, the volume ratio of α-amylase to fermentation broth is 1–1.5:100, the enzymatic hydrolysis conditions are 60℃ for 1–1.5 h, and the pH is 6.5; the volume ratio of alkaline protease to fermentation broth is 2–2.5:100, the enzymatic hydrolysis conditions are 55℃ for 2–2.5 h, and the pH is 9.0; the volume ratio of glucoamylase to fermentation broth is 1–1.5:100, the enzymatic hydrolysis conditions are 50℃ for 2–2.5 h, and the pH is 4.2.

[0016] The present invention also provides a corn husk soluble dietary fiber, which is prepared by the above method.

[0017] The present invention also provides the application of the above method or the above corn husk soluble dietary fiber in the preparation of products that lower blood sugar or help maintain healthy blood sugar levels.

[0018] The beneficial effects of this invention are:

[0019] The Trichoderma reesei TE-36 provided by this invention has a complete cellulase system, fast growth rate, high enzyme production, mild growth conditions, and is suitable for industrial application. Moreover, it can secrete a variety of highly active cell wall hydrolases, such as cellulase and xylanase, which can effectively degrade cellulose and hemicellulose macromolecules in corn husks. When used in the preparation of soluble dietary fiber from corn husks, it can effectively improve the yield, purity, and hypoglycemic activity of soluble dietary fiber from corn husks.

[0020] The method for preparing soluble dietary fiber from corn husks provided by this invention combines the advantages of microbial fermentation and enzymatic hydrolysis through a synergistic fermentation technique, significantly improving hydrolysis efficiency. Furthermore, γ-irradiation technology is used to disrupt the structure of lignocellulose, enhancing enzymatic hydrolysis efficiency and increasing the solubility of water-soluble carbohydrates. The use of ultrafine grinding combined with γ-irradiation further enhances the degradation effect of irradiation. This invention organically integrates multiple treatment methods, not only increasing the yield, purity, and hypoglycemic properties of soluble dietary fiber, but also making it suitable for industrial-scale application. The results of the examples show that the yield of soluble dietary fiber prepared by the method of the present invention is as high as (18.22±0.71)%, and the purity is as high as (86.74±3.56)%. Moreover, when the glucose concentration is 50 mmol / L, the glucose adsorption characteristic of the corn husk soluble dietary fiber prepared by the method of the present invention is 0.93 mmol / g·L; the glucose lag index is 41.97% after 30 min of reaction; the α-glucosidase inhibition rate is 65.50%, and the α-amylase inhibition rate is 21.41%.

[0021] The strain and preparation method proposed in this invention can increase the added value of corn husks, promote the processing and utilization of soluble dietary fiber as a functional product base, and contribute to the development and utilization of agricultural resources, which has important practical significance. Attached Figure Description

[0022] Figure 1 This is a technical roadmap of the preparation method in Example 3;

[0023] Figure 2 Figures showing the yield and purity of soluble dietary fiber from corn husks in different treatment groups;

[0024] Figure 3 This is a graph showing enzyme activity in fermentation broth at different fermentation times;

[0025] Figure 4 A graph showing the glucose adsorption effect of soluble dietary fiber in corn husks;

[0026] Figure 5 A graph showing the effect of glucose absorption retardation index on soluble dietary fiber in corn husks;

[0027] Figure 6 This is a graph showing the inhibitory effect of soluble dietary fiber from corn husks on digestive enzymes.

[0028] Preservation Instructions

[0029] The *Trichoderma reesei* strain TE-36 of this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC), classified and named *Trichoderma reesei*, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 41552, and deposited on October 25, 2024. Detailed Implementation

[0030] This invention provides a *Trichoderma reesei* strain TE-36, which is classified as *Trichoderma reesei* and deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41552. This *Trichoderma reesei* strain TE-36 was isolated, screened, and purified from decaying wood. It possesses a complete cellulase system, exhibits rapid growth, high enzyme production, mild growth conditions, and suitability for industrial applications. It can secrete various highly active cell wall hydrolases, such as cellulase and xylanase, which can effectively degrade cellulose and hemicellulose macromolecules in corn husks.

[0031] The present invention also provides the application of the above-mentioned Trichoderma reesei TE-36 in the preparation of soluble dietary fiber from corn husks.

[0032] The present invention also provides a method for preparing soluble dietary fiber from corn husks, comprising the following steps: mixing corn husk powder with a culture medium to obtain a fermentation culture medium; inoculating the above-mentioned Trichoderma reesei TE-36 seed liquid into the fermentation culture medium for fermentation to obtain a fermentation broth; then sequentially performing enzymatic hydrolysis with α-amylase, alkaline protease and glucoamylase, collecting the supernatant, and precipitating with alcohol to obtain soluble dietary fiber from corn husks.

[0033] This invention does not specifically limit the source of corn husks. The preferred method for preparing the corn husk powder includes the following steps: ultra-finely pulverizing the corn husks and passing them through a 180-200 mesh sieve, then subjecting them to γ-irradiation to obtain the corn husk powder; the intensity of the γ-irradiation is 8-10 kGy. In this invention, the preferred sieve mesh size is 190-200 mesh, and the preferred γ-irradiation intensity is 8.5-9.5 kGy. In this invention, the γ-irradiation time is: from the start of irradiation, stopping when the irradiation intensity reaches 8-10 kGy. This invention uses γ-irradiation technology to disrupt the structure of lignocellulose, improve enzymatic hydrolysis efficiency, and increase the solubility of water-soluble carbohydrates. Furthermore, the combination of ultra-fine pulverization and γ-irradiation treatment can further enhance the degradation effect of the irradiation.

[0034] In this invention, the culture medium preferably comprises 1.0–2.0 g / L K₂HPO₄, 0.3–0.5 g / L MgSO₄·7H₂O, 0.3–0.5 g / L CaCl₂, 0.0015–0.0025 g / L FeSO₄·7H₂O, and 0.0015–0.0025 g / L ZnSO₄·7H₂O; the solvent of the culture medium is preferably water; the concentration of corn husk powder in the fermentation culture medium is preferably 50–66 g / L; after obtaining the fermentation culture medium, it is preferably sterilized before use, and the sterilization method is preferably sterilization at 121°C for 30 min.

[0035] In this invention, the preferred method for obtaining the seed culture of *Trichoderma reesei* TE-36 is as follows: *Trichoderma reesei* TE-36 is inoculated into a seed culture medium and cultured at 25–28°C for 3–4 days; the preferred culture temperature is 26–27°C, and the preferred culture time is 3.5 days. The seed culture medium preferably comprises 25–30 g / L glucose, 2.5–3.0 g / L NaNO3, 0.8–1.0 g / L K2HPO4, 0.5–1.0 g / L KCl, 0.5–1.0 g / L MgSO4·7H2O, and 0.01–0.03 g / L FeSO4. In this invention, the volume ratio of the seed culture to the fermentation culture medium is preferably 8–10:100, more preferably 9:100.

[0036] In this invention, the fermentation preferably includes either microbial fermentation or microbial-enzyme co-fermentation. When microbial fermentation is used, the fermentation temperature is preferably 28–30°C, more preferably 29°C, and the fermentation time is preferably 60–66 h, more preferably 62–64 h. When microbial-enzyme co-fermentation is used, it is preferred to first culture at 28–30°C for 40–42 h, then add β-glucosidase, and continue fermentation for another 20–24 h. In this invention, when using microbial-enzyme co-fermentation, the presence of β-glucosidase can improve the degradation efficiency of cellulose by the entire cellulase complex secreted by *Trichoderma reesei* TE-36, greatly enhancing the hydrolysis efficiency and helping to improve the yield and purity of soluble dietary fiber. In this invention, the volume ratio of β-glucosidase to fermentation medium is preferably 0.4–0.5:100. In this invention, the β-glucosidase is food grade, and the enzyme activity is preferably 1000 U / mL. This invention does not impose specific limitations on the conditions and parameters for the addition of β-glucosidase, because the method and speed of addition vary depending on the type of fermenter. The addition can be carried out according to the actual situation.

[0037] In this invention, after obtaining the fermentation broth, it is preferably sterilized and concentrated. The sterilization conditions are preferably sterilization at 121°C for 30 minutes, and the concentration is preferably reduced to 1 / 3 of the original volume under reduced pressure to obtain a concentrated fermentation broth. Then, soluble dietary fiber is extracted. In this invention, the sequential use of α-amylase, alkaline protease, and glucoamylase for enzymatic hydrolysis means first using α-amylase, then alkaline protease, and finally glucoamylase. After each enzymatic hydrolysis, preferably, enzyme inactivation treatment is performed before proceeding to the next enzyme hydrolysis. In this invention, when using α-amylase for enzymatic hydrolysis, the preferred volume ratio of α-amylase to fermentation broth is 1–1.5:100, more preferably 1.2–1.4:100; the preferred hydrolysis conditions are hydrolysis at 60°C for 1–1.5 h, with a hydrolysis time of 1.1–1.4 h, and a pH of 6.5. When using alkaline protease for enzymatic hydrolysis, the preferred volume ratio of alkaline protease to fermentation broth is 2–2.5:100, more preferably 2.2–2.4:100; the preferred hydrolysis conditions are hydrolysis at 55°C for 2–2.5 h, with a hydrolysis time of 2.1–2.4 h, and a pH of 9.0. When using glucoamylase for enzymatic hydrolysis, the preferred volume ratio of glucoamylase to fermentation broth is 1–1.5:100, more preferably 1.2–1.4:100; the preferred hydrolysis conditions are hydrolysis at 50°C for 2–2.5 h, with a hydrolysis time of 2.1–2.4 h, and a pH of 4.2. This invention does not specifically limit the reagents used for pH adjustment; any reagents conventionally used in the field for pH adjustment can be used. After enzymatic hydrolysis, centrifugation is preferred, and the supernatant is used for alcohol precipitation. The preferred centrifugation conditions are 5000 r / min for 15 min, the preferred ethanol concentration for alcohol precipitation is 95%, and the preferred alcohol precipitation conditions are 4℃ for 10 h. After alcohol precipitation, centrifugation is performed, the precipitate is retained, and after freeze-drying, soluble dietary fiber from corn husks is obtained.

[0038] The present invention also provides a corn husk soluble dietary fiber, which is prepared by the above method.

[0039] This invention also provides the application of the above-described method or the above-described corn husk soluble dietary fiber in the preparation of products that lower blood sugar or help maintain healthy blood sugar levels. In this invention, the type of the product is preferably a drug or food. When the corn husk soluble dietary fiber is used to lower blood sugar, the product is a drug; when the corn husk soluble dietary fiber is used to maintain healthy blood sugar levels, the product is either a food or a drug.

[0040] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0041] Unless otherwise specified, the following embodiments are all conventional methods.

[0042] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0043] The data in the following examples are expressed as mean ± standard deviation.

[0044] Example 1

[0045] A method for preparing soluble dietary fiber from corn husks comprises the following steps:

[0046] (1) The dried corn husk raw material is pulverized by ultra-fine grinding and then passed through an 80-mesh sieve to obtain corn husk powder;

[0047] (2) Preparation of seed culture of Trichoderma reesei TE-36: Take the TE-36PDA slant of Trichoderma reesei, scrape the spores under sterile conditions and put them into sterile water to make the spore concentration 10. 8 The spores were collected at a concentration of 100 g / mL, shaken well, and 5 mL of the spore suspension was inoculated into 100 mL of seed culture medium. The culture was incubated at 28°C for 3 days to obtain the seed culture. The seed culture medium formula was as follows: per 1 L of water, there were 30 g glucose, 3.0 g NaNO3, 1.0 g K2HPO4, 0.5 g KCl, 0.5 g MgSO4·7H2O, and 0.01 g FeSO4.

[0048] (3) Fermentation modification: The corn husk powder obtained in step (1) is mixed with the culture medium to obtain a fermentation culture medium, so that the final fermentation culture medium contains 50g of corn husk powder, 2.0g of K2HPO4, 0.3g of MgSO4·7H2O, 0.3g of CaCl2, 0.0015g of FeSO4·7H2O, and 0.0015g of ZnSO4·7H2O per 1L of water; sterilize at 121℃ for 30min; after cooling, inoculate the seed liquid of Trichoderma reesei TE-36 obtained in step (2) at a volume ratio of 10:100 between the seed liquid and the fermentation culture medium, and culture at 30℃ for 62h to obtain a fermentation liquid, then sterilize at 121℃ for 30min, and after cooling, concentrate under reduced pressure to 1 / 3 of the original fermentation liquid volume to obtain a fermentation concentrate;

[0049] (4) Extraction of dietary fiber: Adjust the pH of the fermentation concentrate obtained in step (3) to 6.5, and add α-amylase at a volume ratio of 1:100 (the enzyme activity of the α-amylase is 2×10⁻⁶). 5 The enzyme was inactivated by enzymatic hydrolysis at 60℃ for 1 hour followed by boiling in a water bath. After the temperature dropped to 55℃, the pH of the solution was adjusted to 9.0. Alkaline protease (with an activity of 2 × 10⁻⁶ U / g) was then added at a volume ratio of 2:100 (the volume ratio of alkaline protease to fermentation concentrate was 2:100). 5The enzyme was hydrolyzed at 55℃ for 2 hours (U / g), then heated to inactivate the enzyme. The pH of the solution was adjusted back to 4.2, and glucoamylase (with an activity of 1.5 × 10⁻⁶ U / g) was added at a volume ratio of 1:100 to the fermentation concentrate. 5 The enzyme was enzymatically hydrolyzed at 50℃ for 2 hours (U / g) and then inactivated again. After cooling, the solution was centrifuged at 5000 rpm for 15 minutes, and the supernatant was concentrated to 1 / 3 of its original volume under vacuum. Then, 4 volumes of 95% ethanol were added, and the solution was precipitated at 4℃ for 10 hours. The precipitate was centrifuged at 5000 rpm for 15 minutes and retained. The precipitate was then frozen at -60℃ until dried to obtain corn husk soluble dietary fiber.

[0050] The experiment was repeated three times. The yield of soluble dietary fiber obtained in this example (the yield is the percentage of the mass of soluble dietary fiber to the mass of corn husk raw material) was (11.86±0.39)%, and the purity (the purity is the percentage of the mass of the sample after deducting the mass of starch, protein and ash) was (78.38±2.46)%.

[0051] Example 2

[0052] The difference from Example 1 is that in step (3), after inoculating the seed culture, the culture is first incubated at 30°C for 42 hours, and then β-glucosidase (the volume ratio of β-glucosidase to fermentation medium is 0.4:100) is added, and fermentation continues for 20 hours to obtain the fermentation broth. The rest is the same as in Example 1.

[0053] The results showed that the yield of soluble dietary fiber obtained in this example was (13.64±0.42)%, and the purity was (82.39±2.78)%.

[0054] Example 3

[0055] The difference from Example 2 is that step (1) is as follows: the dried corn husk raw material is ultra-finely pulverized and passed through a 200-mesh sieve, and then subjected to γ-irradiation at an intensity of 8 kGy to obtain corn husk powder. The rest is the same as in Example 2. The technical route diagram of the preparation method in this example is as follows: Figure 1 As shown, the specific steps are as follows:

[0056] (1) The dried corn husk raw material was ultra-finely pulverized and passed through a 200-mesh sieve, and then subjected to γ-irradiation with an irradiation intensity of 8 kGy to obtain corn husk powder;

[0057] (2) Preparation of seed culture of Trichoderma reesei TE-36: Take the TE-36PDA slant of Trichoderma reesei, scrape the spores under sterile conditions and put them into sterile water to make the spore concentration 10. 8The spores were collected at a concentration of 100 g / mL, shaken well, and 5 mL of the spore suspension was inoculated into 100 mL of seed culture medium. The culture was incubated at 28°C for 3 days to obtain the seed culture. The seed culture medium formula was as follows: per 1 L of water, there were 30 g glucose, 3.0 g NaNO3, 1.0 g K2HPO4, 0.5 g KCl, 0.5 g MgSO4·7H2O, and 0.01 g FeSO4.

[0058] (3) Fermentation modification: The corn husk powder obtained in step (1) is mixed with the culture medium to obtain a fermentation culture medium, so that the final fermentation culture medium contains 50g of corn husk powder, 2.0g of K2HPO4, 0.3g of MgSO4·7H2O, 0.3g of CaCl2, 0.0015g of FeSO4·7H2O, and 0.0015g of ZnSO4·7H2O per 1L of water; sterilize at 121℃ for 30min; after cooling, inoculate the seed liquid of Trichoderma reesei TE-36 obtained in step (2) at a volume ratio of 10:100 between the seed liquid and the fermentation culture medium, first culture at 30℃ for 42h, then add β-glucosidase (the volume ratio of β-glucosidase to the fermentation culture medium is 0.4:100), continue fermentation for 20h to obtain a fermentation broth, then sterilize at 121℃ for 30min, cool and concentrate under reduced pressure to 1 / 3 of the original fermentation broth volume to obtain a fermentation concentrate;

[0059] (4) Extraction of dietary fiber: Adjust the pH of the fermentation concentrate obtained in step (3) to 6.5, and add α-amylase at a volume ratio of 1:100 (the enzyme activity of the α-amylase is 2×10⁻⁶). 5 The enzyme was inactivated by enzymatic hydrolysis at 60℃ for 1 hour followed by boiling in a water bath. After the temperature dropped to 55℃, the pH of the solution was adjusted to 9.0. Alkaline protease (with an activity of 2 × 10⁻⁶ U / g) was then added at a volume ratio of 2:100 (the volume ratio of alkaline protease to fermentation concentrate was 2:100). 5 The enzyme was hydrolyzed at 55℃ for 2 hours (U / g), then heated to inactivate the enzyme. The pH of the solution was adjusted back to 4.2, and glucoamylase (with an activity of 1.5 × 10⁻⁶ U / g) was added at a volume ratio of 1:100 to the fermentation concentrate. 5 The enzyme was hydrolyzed at 50℃ for 2 hours (U / g) and then inactivated again. After cooling, the solution was centrifuged at 5000 rpm for 15 minutes, and the supernatant was concentrated to 1 / 3 of its original volume under vacuum. Then, 4 times the volume of 95% ethanol was added, and the solution was precipitated at 4℃ for 10 hours. The precipitate was centrifuged and retained, and then freeze-dried to obtain corn husk soluble dietary fiber.

[0060] The experiment was repeated three times, and the results showed that the yield of soluble dietary fiber obtained in this example was (18.22±0.71)%, and the purity was (86.74±3.56)%.

[0061] Comparative Example 1

[0062] The difference from Example 1 is that it does not contain steps (2) and (3). After obtaining corn husk powder, water is added until 50g of corn husk powder is contained in every 1L of water to obtain corn husk powder solution. Then, the sequential enzymatic hydrolysis of step (4) is performed. The fermentation concentrate in step (4) is replaced with corn husk powder solution. The rest is the same as in Example 1.

[0063] The results showed that the yield of soluble dietary fiber in this comparative group was (3.53±0.08)%, and the purity was (69.31±1.23)%.

[0064] Comparative Example 2

[0065] The difference from Comparative Example 1 is that step (1) involves pulverizing the dried corn husk raw material into ultrafine powder, passing it through a 200-mesh sieve, and then subjecting it to γ-irradiation with an intensity of 8 kGy to obtain corn husk powder. The rest is the same as Comparative Example 1.

[0066] The results showed that the yield of soluble dietary fiber in this comparative group was (6.28±0.31)%, and the purity was (75.53±2.21)%.

[0067] Example 1 was designated as the fermentation group, Example 2 as the bacterial-enzyme co-fermentation group, and Example 3 as the γ-irradiation combined with bacterial-enzyme co-fermentation group. Comparative Example 1 was designated as the control group, and Comparative Example 2 as the γ-irradiation group. The comparison results of the yield and purity of soluble dietary fiber obtained from Examples 1-3 and Comparative Examples 1-2 are shown in the figure. Figure 2 .

[0068] Example 4

[0069] The activities of cellulase and β-glucosidase in the fermentation broth at different fermentation times in Example 3 were determined:

[0070] Cellulase activity assay: Take 0.2 mL of fermentation broth, add 1.8 mL of sodium carboxymethyl cellulose solution (mass fraction 1%), and incubate at 50℃ for 30 min. For the blank control group, add 0.2 mL of fermentation broth and 1.8 mL of acetate buffer (pH 4.8), and incubate at 50℃ for 30 min. Then, take 2 mL of 3,5-dinitrosalicylic acid test solution, shake well, and incubate in a boiling water bath for 10 min. After cooling, add water to make up to 15 mL, zero the chamber using the blank control, and measure the OD value at 550 nm. One unit of sodium carboxymethyl cellulose enzyme activity is defined as the amount of enzyme that hydrolyzes 1% sodium carboxymethyl cellulose solution per minute to produce 1 μg of reducing sugar under the conditions of 50℃ and pH 4.8.

[0071] β-glucosidase activity assay: The assay was performed strictly according to the kit (Sigma, MAK129). The substrate was p-nitrophenyl-β-D-glucopyranoside. The amount of p-nitrophenol generated after 10 min of reaction was measured at 405 nm using a spectrophotometer. The amount of enzyme releasing p-nitrophenol at a rate of 1 μmol / min was defined as the international unit (IU) of β-glucosidase activity.

[0072] The results are as follows Figure 3 As shown, the addition of β-glucosidase during fermentation effectively increases cellulase activity, thereby enhancing the degradation efficiency of cellulose by the entire cellulase complex secreted by *Trichoderma reesei* TE-36, significantly improving hydrolysis efficiency. Figure 3 It can be seen that adding β-glucosidase at 42 hours of fermentation yields the best results.

[0073] Example 5

[0074] The glucose adsorption capacity of corn husk soluble dietary fiber obtained in Example 3 (referred to as combined modified SDF) and Comparative Example 1 (referred to as unmodified SDF) was determined respectively.

[0075] Take 0.5g of the soluble dietary fiber sample obtained in Example 3 or Comparative Example 1 and mix it with 50mL of glucose solutions of different concentrations (10, 20, 35, 50mmol / L). Shake at 37℃ for 6h. Centrifuge at 4500r / min for 10min, collect the supernatant, and determine the glucose content using a kit method.

[0076]

[0077] The results are as follows Figure 4 As shown, the glucose adsorption capacity of corn husk soluble dietary fiber increases with increasing glucose concentration. When the glucose concentration is 50 mmol / L, the glucose adsorption capacity of corn husk soluble dietary fiber obtained in Example 3 of this invention is 0.93 mmol / g·L, and the glucose adsorption capacity obtained in Comparative Example 1 is 0.73 mmol / g·L.

[0078] Example 6

[0079] The glucose lag index of corn husk soluble dietary fiber obtained in Example 3 (referred to as combined modified SDF) and Comparative Example 1 (referred to as unmodified SDF) was determined respectively.

[0080] Different groups of soluble dietary fiber were dissolved in 50 mmol / L glucose solution at a ratio of 1 g: 50 mL. The solution was placed in a dialysis bag with a molecular weight cutoff of 6000 Da, placed in a beaker, and dialyzed at 37 °C using 4 times the volume of distilled water as the dialysate. The glucose content of the dialysate was measured at 30, 60, 90, and 120 min.

[0081]

[0082] The results are as follows Figure 5 As shown, with the increase of reaction time, the glucose lag index of corn husk soluble dietary fiber showed a trend of first increasing and then decreasing. When the reaction time was 60 min, the glucose lag index of corn husk soluble dietary fiber obtained in Example 3 reached the maximum value of 41.97%, while the glucose lag index of Comparative Example 1 was 11.24%.

[0083] Example 7

[0084] The inhibitory activity of corn husk soluble dietary fiber obtained in Example 3 (referred to as combined modified SDF) and Comparative Example 1 (referred to as unmodified SDF) on digestive enzymes was determined respectively.

[0085] Assay for α-glucosidase inhibitory activity: 0.5 mL of α-glucosidase (0.4 U / mL) and 1.0 mL of PBS (pH 6.8) were mixed with 20 mg of soluble dietary fiber. The mixture was incubated at 37 °C for 15 min. Then, 1.0 mL of 10 mmol / L p-nitrophenyl-β-D-galactopyranoside was added as a substrate to initiate the reaction. After 30 min, 5.0 mL of 0.2 mol / L Na₂CO₃ was added to immediately terminate the reaction. The reaction was centrifuged at 4500 r / min for 10 min, and the supernatant was retained. The absorbance was measured at 405 nm.

[0086] Assay for α-amylase activity: 0.5 mL of α-amylase (6 U / mL) was mixed with 1.0 mL of PBS (pH 6.9) and 20 mg of soluble dietary fiber, and incubated at 37 °C for 15 min. 1.0 mL of 1% soluble starch solution was added as substrate. After reacting for 5 min, 5.0 mL of 3,5-dinitrosalicylic acid reagent was added, and the mixture was boiled for 10 min. After cooling, the mixture was centrifuged at 4500 rpm for 10 min. The supernatant was retained, and its absorbance was measured at 500 nm.

[0087] The inhibition rates of α-glucosidase and α-amylase mentioned above are calculated using the following formula: The digestive enzymes in the formula refer to α-glucosidase or α-amylase:

[0088]

[0089] In the formula, A1 is the absorbance of the group without added digestive enzymes; A2 is the absorbance of the group without dietary fiber; A3 is the absorbance of the group with added dietary fiber and digestive enzymes; and A4 is the absorbance of the group with added dietary fiber but no added digestive enzymes.

[0090] The results are as follows Figure 6 As shown, the α-glucosidase inhibition rate of the corn husk soluble dietary fiber obtained in Example 3 of the present invention was 65.50%, and the α-amylase inhibition rate was 21.41%, while the α-glucosidase inhibition rate of Comparative Example 1 was 32.45%, and the α-amylase inhibition rate was 8.5%.

[0091] Example 8

[0092] A method for preparing soluble dietary fiber from corn husks comprises the following steps:

[0093] (1) The dried corn husk raw material was ultra-finely pulverized and passed through a 190-mesh sieve, and then subjected to γ-irradiation with an irradiation intensity of 10 kGy to obtain corn husk powder.

[0094] (2) Preparation of seed culture of Trichoderma reesei TE-36: Take the TE-36PDA slant of Trichoderma reesei, scrape the spores under sterile conditions and put them into sterile water to make the spore concentration 10. 8 The spores were collected at a concentration of 100 g / mL, shaken well, and 5 mL of the spore suspension was inoculated into 100 mL of seed culture medium. The culture was incubated at 26 °C for 4 days to obtain the seed culture. The seed culture medium formula was as follows: per 1 L of water, there were 25 g of glucose, 2.5 g of NaNO3, 0.8 g of K2HPO4, 1.0 g of KCl, 1.0 g of MgSO4·7H2O, and 0.03 g of FeSO4.

[0095] (3) Fermentation modification: The corn husk powder obtained in step (1) is mixed with the culture medium to obtain a fermentation culture medium, so that the final fermentation culture medium contains 66g of corn husk powder, 1.0g of K2HPO4, 0.5g of MgSO4·7H2O, 0.5g of CaCl2, 0.0025g of FeSO4·7H2O, and 0.0025g of ZnSO4·7H2O per 1L of water; sterilize at 121℃ for 30min; after cooling, inoculate the seed liquid of Trichoderma reesei TE-36 obtained in step (2) at a volume ratio of 8:100 between the seed liquid and the fermentation culture medium, first culture at 28℃ for 40h, then add β-glucosidase (the volume ratio of β-glucosidase to the fermentation culture medium is 0.5:100), continue fermentation for 24h to obtain a fermentation broth, then sterilize at 121℃ for 30min, cool and concentrate under reduced pressure to 1 / 3 of the original fermentation broth volume to obtain a fermentation concentrate;

[0096] (4) Extraction of dietary fiber: Adjust the pH of the fermentation concentrate obtained in step (3) to 6.5, and add α-amylase at a volume ratio of 1.4:100 (the enzyme activity of the α-amylase is 2×10⁻⁶). 5 The enzyme was inactivated by enzymatic hydrolysis at 60℃ for 1.2 h followed by boiling in a water bath. After the temperature dropped to 55℃, the pH of the solution was adjusted to 9.0. Alkaline protease (with an activity of 2 × 10⁻⁶ U / g) was then added at a volume ratio of 2.3:100 to the fermentation concentrate. 5 The enzyme was hydrolyzed at 55℃ for 2.2 hours (U / g), then heated to inactivate the enzyme. The pH of the solution was adjusted back to 4.2, and glucoamylase (with an activity of 1.5 × 10⁻⁶ U / g) was added at a volume ratio of 1.3:100 to the fermentation concentrate. 5 The enzyme was hydrolyzed at 50℃ for 2.4 h, and then inactivated again. After cooling, the solution was centrifuged at 5000 r / min for 15 min, and the supernatant was concentrated to 1 / 3 of its original volume under vacuum. Then, 4 volumes of 95% ethanol were added and precipitated at 4℃ for 10 h. The precipitate was centrifuged and retained, and then freeze-dried to obtain corn husk soluble dietary fiber.

[0097] Testing and verification showed that the yield and purity of the corn husk soluble dietary fiber obtained in this example were higher than those in comparative examples 1 and 2, and the inhibition rates of α-glucosidase and α-amylase were similar to those in example 3.

[0098] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing corn bran soluble dietary fiber, characterized by, The method comprises the following steps: mixing corn bran powder with culture medium to obtain fermentation culture medium; inoculating seed liquid of Trichoderma reesei TE-36 into the fermentation culture medium to carry out fermentation, and obtaining fermentation liquor; and then sequentially carrying out enzymolysis by using α-amylase, alkaline protease and glucoamylase, collecting supernatant, and alcohol precipitation to obtain corn bran soluble dietary fiber. The Trichoderma reesei fungus TE-36 is named as Trichoderma reesei Trichoderma reesei and is preserved in the China General Microbiological Culture Collection Center with a preservation number of CGMCC No. 41552. The preparation method of the corn bran powder comprises the following steps: after corn bran is ultra-finely pulverized, the corn bran is passed through a 180-200 mesh sieve, and then γ-irradiation is adopted to obtain the corn bran powder; the intensity of the γ-irradiation is 8-10 kGy. The fermentation is carried out at 28-30 ℃ for 40-42 h, then β-glucosidase is added, and the fermentation is continued for 20-24 h; the volume ratio of the β-glucosidase to the fermentation culture medium is 0.4-0.5:

100.

2. The method of claim 1, wherein, The culture medium comprises K2HPO4 1.0-2.0 g / L, MgSO4·7H2O 0.3-0.5 g / L, CaCl2 0.3-0.5 g / L, FeSO4·7H2O 0.0015-0.0025 g / L and ZnSO4·7H2O 0.0015-0.0025 g / L; and the concentration of the corn bran powder in the fermentation culture medium is 50-66 g / L.

3. The method of claim 1, wherein, The seed liquid is obtained by inoculating Trichoderma reesei TE-36 into seed culture medium and culturing at 25-28 ℃ for 3-4 days; the seed culture medium comprises glucose 25-30 g / L, NaNO3 2.5-3.0 g / L, K2HPO4 0.8-1.0 g / L, KCl 0.5-1.0 g / L, MgSO4·7H2O 0.5-1.0 g / L and FeSO4 0.01-0.03 g / L; and the volume ratio of the seed liquid to the fermentation culture medium is 8-10:

100.

4. The method of claim 1, wherein, The volume ratio of the α-amylase to the fermentation liquor is 1-1.5:100, the enzymolysis condition is 60 ℃ for 1-1.5 h, and the pH is 6.5; the volume ratio of the alkaline protease to the fermentation liquor is 2-2.5:100, the enzymolysis condition is 55 ℃ for 2-2.5 h, and the pH is 9.0; and the volume ratio of the glucoamylase to the fermentation liquor is 1-1.5:100, the enzymolysis condition is 50 ℃ for 2-2.5 h, and the pH is 4.

2.

5. A corn bran soluble dietary fiber, characterized by, The corn bran soluble dietary fiber is prepared by the method of any one of claims 1-4.

6. Use of the method of any one of claims 1-4 or the corn bran soluble dietary fiber of claim 5 in the preparation of a product for reducing blood sugar or helping to maintain a healthy level of blood sugar.

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