Rice bran feruloyl oligosaccharide as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to rice bran feruloyl oligosaccharide as well as a preparation method and application thereof. The invention provides a preparation method of rice bran feruloyl oligosaccharide, which comprises the following steps: mixing and degreasing rice bran and n-hexane, and performing gelatinization and enzymolysis to obtain rice bran insoluble dietary fiber; the rice bran insoluble dietary fibers are subjected to enzymolysis, and supernate is taken after centrifugation; and carrying out gradient alcohol precipitation on the supernate by using 50% ethanol, 70% ethanol and 90% ethanol in sequence to obtain the rice bran feruloyl oligosaccharide. The prepared rice bran feruloyl oligosaccharide has the effect of inhibiting the activity of alpha-amylase and alpha-glucosidase, and can be used as a food-borne hypoglycemic component for developing functional foods, medicines and the like for regulating blood sugar.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a rice bran ferulic acid oligosaccharide, its preparation method, and its application. Background Technology

[0002] Diabetes mellitus is a global metabolic disease characterized by chronic hyperglycemia, with its incidence and mortality rates rising annually, posing a serious threat to human health. Controlling postprandial blood glucose is a key aspect of diabetes management. Alpha-amylase hydrolyzes the α-1,4-glycosidic bonds within starch, degrading polysaccharides into oligosaccharides; α-glucosidase is responsible for breaking down oligosaccharides and disaccharides into absorbable monosaccharides, directly leading to a rapid rise in postprandial blood glucose. Therefore, inhibiting the activity of digestive enzymes and delaying the digestion and absorption of carbohydrates is considered one of the important strategies for effectively controlling postprandial blood glucose.

[0003] Currently, widely used digestive enzyme inhibitors in clinical practice, such as acarbose and voglibose, while showing definite efficacy, often cause gastrointestinal side effects such as bloating, flatulence, and diarrhea, which limits their clinical application to some extent. Therefore, finding highly effective and low-toxicity digestive enzyme inhibitors from natural resources has become a hot topic in current drug development and functional food development.

[0004] Rice bran is a major byproduct of rice processing, rich in protein, lipids, vitamins, and various bioactive substances. For a long time, rice bran has been primarily used as animal feed or discarded, its high added value not being fully exploited, resulting in significant resource waste. Polysaccharides, as important biomolecules, are widely found in plants, animals, and microorganisms, and have attracted considerable attention due to their various biological activities, including immunomodulation, antitumor activity, antioxidant activity, and hypoglycemic activity. In recent years, studies have reported that polysaccharides extracted from rice bran possess certain biological activities, such as antioxidant and immune-enhancing activities. However, current research on rice bran polysaccharides largely focuses on conventional extraction processes and basic physiological functions, with limited and in-depth research on their specific inhibition of α-amylase and α-glucosidase activity. In particular, there is currently a lack of clear technical guidance on how to obtain rice bran polysaccharide components with significantly enhanced α-glucosidase inhibitory activity through specific and optimized preparation methods (such as extraction, separation, and purification processes). The inhibitory activity of rice bran polysaccharides obtained using existing technologies is often insufficient, limiting their application in hypoglycemic functional foods or drugs.

[0005] Therefore, there is an urgent need in this field for a product that can be specifically prepared to have high inhibitory activity against digestive enzymes, and for its preparation method and application to fill the gap in the existing technology, realize the high-value utilization of rice bran resources, and provide a new potential solution derived from natural products for the prevention and treatment of diabetes. Summary of the Invention

[0006] The purpose of this invention is to provide a rice bran feruloyl oligosaccharide, its preparation method and application. The rice bran feruloyl oligosaccharide can significantly inhibit the activity of α-amylase and α-glucosidase.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing rice bran ferulic acid oligosaccharides, comprising the following steps: (1) Mix rice bran and n-hexane to defatt the rice, and obtain defatted rice bran; (2) The defatted rice bran was gelatinized and enzymatically hydrolyzed to obtain insoluble dietary fiber from rice bran; (3) Enzymatically hydrolyze and centrifuge the insoluble dietary fiber of rice bran, and take the supernatant; use 50% ethanol, 70% ethanol and 90% ethanol to perform gradient alcohol precipitation of the supernatant to obtain rice bran feruloyl oligosaccharide.

[0008] Preferably, in step (1), the mass-to-volume ratio of the rice bran and n-hexane is 250 mg: 450~550 mL; the degreasing time is 12~14 h; the degreasing process requires stirring at a speed of 160~180 rpm; and the degreasing is performed 1~3 times.

[0009] Preferably, in step (2), the gelatinization step is as follows: mix defatted rice bran and water, adjust the pH to 5.5~6.5, and gelatinize at 90~100℃ for 8~12 minutes to obtain gelatinized defatted rice bran; The mass-to-volume ratio of the defatted rice bran and water is 50 mg: 450~550 mL.

[0010] Preferably, in step (2), the enzymatic hydrolysis includes the following steps: mixing the gelatinized defatted rice bran with heat-resistant α-amylase, alkaline protease and amyloglucosidase in sequence for enzymatic hydrolysis.

[0011] Preferably, the amount of the heat-resistant α-amylase added is 4% of the mass of defatted rice bran; the enzymatic hydrolysis conditions of the defatted rice bran and the heat-resistant α-amylase are: enzymatic hydrolysis at 90~100℃ for 25~35 min; The amount of alkaline protease added is 2% of the mass of defatted rice bran; the enzymatic hydrolysis conditions of the alkaline protease are: adjust the pH to 8-9, and hydrolyze at 45-55℃ for 1.5-2.5 hours. The amount of amyloglucosidase added is 2% of the mass of defatted rice bran; the enzymatic hydrolysis conditions of the amyloglucosidase are: adjust the pH to 4-5, and hydrolyze at 50-60℃ for 0.5-1.5h.

[0012] Preferably, in step (3), the enzymatic hydrolysis step is: enzymatically hydrolyzing rice bran insoluble dietary fiber and xylanase solution at 50~60℃ for 12~14h; The mass-to-volume ratio of the rice bran insoluble dietary fiber to the xylanase solution is 100 mg: 1000 mL.

[0013] The present invention also provides the preparation method described above for preparing rice bran ferulic acid oligosaccharides.

[0014] The present invention also provides the use of rice bran ferulic acid oligosaccharide prepared by the preparation method described above, or the use of rice bran ferulic acid oligosaccharide described above in the preparation of products that inhibit the activity of α-amylase and α-glucosidase.

[0015] The present invention also provides the rice bran feruloyl oligosaccharide prepared by the preparation method described above, or the application of the rice bran feruloyl oligosaccharide described above in the preparation of products for regulating blood sugar.

[0016] The present invention also provides the rice bran feruloyl oligosaccharide prepared by the preparation method described above, or the application of the rice bran feruloyl oligosaccharide described above in the preparation of products for treating diabetes.

[0017] Beneficial effects: Compared with conventionally extracted rice bran polysaccharides, the rice bran feruloyl oligosaccharide extracted by enzymatic hydrolysis followed by alcohol precipitation in this invention has a significant inhibitory effect on α-amylase and α-glucosidase activity, and its effect is comparable to that of acarbose. However, because it is derived from a natural grain by-product, it is expected to have better biocompatibility and safety. It can be used as a food-derived hypoglycemic component for the development of functional foods, medicines, etc., for the purpose of regulating blood sugar, providing a new material basis for the development of novel, low-toxicity natural inhibitors of postprandial hyperglycemia.

[0018] This invention transforms rice bran, which is usually used as feed or waste, into a high-value-added bioactive raw material, greatly enhancing the economic value of rice processing by-products. Attached Figure Description

[0019] Figure 1 The chromatogram shows the molecular weight distribution of rice bran ferulic acid oligosaccharides. Figure 2 Liquid chromatogram of the monosaccharide composition of rice bran ferulic acid oligosaccharides; Figure 3 The ultraviolet spectrum of rice bran feruloyl oligosaccharide; Figure 4 Fourier transform infrared spectrum of rice bran ferulic acid oligosaccharide; Figure 5 The inhibition rate and IC50 of rice bran feruloyl oligosaccharides against α-amylase activity 50 value; Figure 6 The inhibition rate and IC50 of rice bran feruloyl oligosaccharides on α-glucosidase activity 50 value; Figure 7 Lineweaver-Burk plots showing the inhibition of α-amylase and α-glucosidase by rice bran ferulic oligosaccharides, where A represents α-amylase and B represents α-glucosidase. Detailed Implementation

[0020] This invention provides a method for preparing rice bran ferulic acid oligosaccharides, comprising the following steps: (1) Mix rice bran and n-hexane to defatt the rice, and obtain defatted rice bran; (2) The defatted rice bran was gelatinized and enzymatically hydrolyzed to obtain insoluble dietary fiber from rice bran; (3) Enzymatically hydrolyze and centrifuge the insoluble dietary fiber of rice bran, and take the supernatant; use 50% ethanol, 70% ethanol and 90% ethanol to perform gradient alcohol precipitation of the supernatant to obtain rice bran feruloyl oligosaccharide.

[0021] In this invention, in step (1), the mass-to-volume ratio of the rice bran and n-hexane is 250mg:450~550mL, preferably 250mg:480~520mL, and more preferably 250mg:500mL; the degreasing time is 12~14h, preferably 13h; the degreasing process requires stirring, and the stirring speed is 160~180rpm, preferably 165~175rpm, and more preferably 170rpm; the degreasing is performed 1~3 times, preferably 2 times.

[0022] In this invention, in step (2), the gelatinization step is as follows: the defatted rice bran and water are mixed, the pH is adjusted to 5.5~6.5, and then gelatinized at 90~100℃ for 8~12 minutes to obtain the gelatinized defatted rice bran; The mass-to-volume ratio of the defatted rice bran to water is 50 mg: 450-550 mL, preferably 50 mg: 480-520 mL, and more preferably 50 mg: 500 mL; The pH is preferably 6.0; the gelatinization temperature is preferably 93~97℃, more preferably 95℃; the gelatinization time is preferably 9~11min, more preferably 10min.

[0023] The enzymatic hydrolysis includes the following steps: the gelatinized defatted rice bran is sequentially mixed with heat-resistant α-amylase, alkaline protease and amyloglucosidase for enzymatic hydrolysis.

[0024] The amount of heat-resistant α-amylase added is 4% (w / w) of the defatted rice bran mass; the enzymatic hydrolysis conditions of the defatted rice bran and heat-resistant α-amylase are: enzymatic hydrolysis at 90~100℃ for 25~35 min; the hydrolysis temperature is preferably 93~97℃, more preferably 95℃; the hydrolysis time is preferably 28~32 min, more preferably 30 min; the heat-resistant α-amylase is used to remove protein from the defatted rice bran; The amount of alkaline protease added is 2% (w / w) of the defatted rice bran mass; the enzymatic hydrolysis conditions of the alkaline protease are: adjusting the pH to 8-9, and hydrolyzing at 45-55℃ for 1.5-2.5 h; the preferred pH for hydrolysis is 8.5; the preferred hydrolysis time is 48-52℃, more preferably 50℃; the preferred hydrolysis time is 1.8-2.2 h, more preferably 2.0 h; the alkaline protease is used to remove starch. The amount of amyloglucosidase added is 2% (w / w) of the defatted rice bran mass; the enzymatic hydrolysis conditions of the amyloglucosidase are: adjusting the pH to 4-5, and hydrolyzing at 50-60℃ for 0.5-1.5 h; the preferred pH for hydrolysis is 4.5; the preferred hydrolysis time is 53-57℃, more preferably 55℃; the preferred hydrolysis time is 0.8-1.2 h, more preferably 1.0 h; the amyloglucosidase is used to further degrade the residual starch. The enzymatic hydrolysis process also includes the following steps: after inactivating the enzyme by boiling in a water bath for 10 minutes, centrifuge at 6000 rpm for 15 minutes to collect the precipitate, wash the precipitate with deionized water until the supernatant is free of white flocculent matter; dry the washed precipitate at 50°C for 24 hours to obtain rice bran insoluble dietary fiber.

[0025] In this invention, in step (3), the rice bran insoluble dietary fiber needs to be pulverized and passed through an 80-mesh sieve before enzymatic hydrolysis; The enzymatic hydrolysis step is as follows: insoluble dietary fiber from rice bran and xylanase solution are enzymatically hydrolyzed at 50-60℃ for 12-14 hours; The enzymatic hydrolysis temperature is preferably 53~57℃, more preferably 55℃; the enzymatic hydrolysis time is preferably 13h. The mass-to-volume ratio of the rice bran insoluble dietary fiber and xylanase solution is 100 mg: 1000 mL; The xylanase solution was prepared by mixing xylanase and acetate buffer. The mass-to-volume ratio of xylanase to acetate buffer is 1 mg: 80-120 mL, preferably 1 mg: 90-110 mL, and more preferably 1 mg: 100 mL; The pH of the acetic acid buffer solution is 5.0; The enzyme needs to be inactivated by boiling water bath for 10 minutes after enzymatic hydrolysis. The centrifugation speed is 5500~6500 rpm, preferably 5800~6200 rpm, more preferably 6000 rpm, and the centrifugation time is 15~25 min, preferably 18~22 min, more preferably 20 min; The precipitate obtained after gradient alcohol precipitation needs to be reconstituted with distilled water, and the residual ethanol is then evaporated by rotary evaporation at 45°C and freeze-dried to obtain rice bran ferulic acid oligosaccharide.

[0026] The present invention also provides the preparation method described above for preparing rice bran ferulic acid oligosaccharides.

[0027] The present invention also provides the use of rice bran ferulic acid oligosaccharide prepared by the preparation method described above, or the use of rice bran ferulic acid oligosaccharide described above in the preparation of products that inhibit the activity of α-amylase and α-glucosidase.

[0028] The present invention also provides the rice bran feruloyl oligosaccharide prepared by the preparation method described above, or the application of the rice bran feruloyl oligosaccharide described above in the preparation of products for regulating blood sugar.

[0029] The present invention also provides the rice bran feruloyl oligosaccharide prepared by the preparation method described above, or the application of the rice bran feruloyl oligosaccharide described above in the preparation of products for treating diabetes.

[0030] 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.

[0031] The CAS numbers of the enzymes used in the embodiments and comparative examples of this invention are as follows: Thermoresistant α-amylase: 9001-19-8; Alkaline protease: 9014-01-1; Amyloglucosidase: 9032-08-0; Xylanase: 9025-57-4.

[0032] Example 1: A rice bran ferulic acid oligosaccharide

[0033] (1) 250g of fresh rice bran and 500mL of n-hexane were stirred and mixed at 170rpm for 12h to degrease the rice bran. The degreasing process was repeated twice to obtain degreased rice bran. (2) Mix 50g defatted rice bran with 500mL distilled water, adjust the pH to 6.0, gelatinize in a 95℃ water bath for 10min, add 4% (w / w) heat-resistant α-amylase of defatted rice bran for starch hydrolysis for 30min; then adjust the pH to 8.5, add 2% (w / w) alkaline protease of defatted rice bran, stir in a 50℃ water bath for 2h to remove protein; then adjust the pH to 4.5, add 2% (w / w) amyloglucosidase of defatted rice bran, stir in a 55℃ water bath for 1h to further degrade residual starch, and inactivate the enzyme in a boiling water bath for 10min; after cooling to 25℃, centrifuge at 6000rpm for 15min to collect the precipitate, wash the precipitate with deionized water until the supernatant is free of white flocculent matter; dry the washed precipitate at 50℃ for 24h to obtain rice bran insoluble dietary fiber; (3) After the rice bran insoluble dietary fiber was pulverized and passed through an 80-mesh sieve, xylanase solution (xylanase and acetate buffer were mixed at a mass-volume ratio of 100g:1000mL) was added. The mixture was enzymatically hydrolyzed in a constant temperature water bath at 55℃ for 12h, and then the enzyme was inactivated by boiling in a water bath for 10min. After cooling to 25℃, the supernatant was collected by centrifugation at 6000rpm for 20min. The supernatant was precipitated with 50% ethanol, and the supernatant was then precipitated again with 70% ethanol. The supernatant was then further precipitated with 90% ethanol. The final precipitate was reconstituted with distilled water, and the residual ethanol was evaporated by rotary evaporation at 45℃. The mixture was then freeze-dried to obtain rice bran ferulic oligosaccharide, which was named 90%RBFOs.

[0034] Example 2: A rice bran ferulic acid oligosaccharide

[0035] (1) 250g of fresh rice bran and 450mL of n-hexane were stirred and mixed at 160rpm for 14h to degrease the rice bran. The degreasing process was repeated twice to obtain degreased rice bran. (2) Mix 50g of defatted rice bran with 550mL of distilled water, adjust the pH to 5.5, gelatinize in a 100℃ water bath for 8min, add 4% (w / w) of heat-resistant α-amylase of defatted rice bran for starch hydrolysis for 25min; then adjust the pH to 8, add 2% (w / w) of alkaline protease of defatted rice bran, stir in a 55℃ water bath for 1.5h to remove protein; then adjust the pH to 5, add 2% (w / w) of amyloglucosidase of defatted rice bran, stir in a 60℃ water bath for 1.5h to further degrade residual starch, inactivate enzyme in a boiling water bath for 10min; after cooling to 25℃, centrifuge at 6000rpm for 15min to collect the precipitate, wash the precipitate with deionized water until the supernatant is free of white flocculent matter; dry the washed precipitate at 50℃ for 24h to obtain rice bran insoluble dietary fiber; (3) After the rice bran insoluble dietary fiber was pulverized and passed through an 80-mesh sieve, xylanase solution (xylanase and acetate buffer were mixed at a mass-volume ratio of 100g:1000mL) was added. The mixture was then hydrolyzed in a constant temperature water bath at 50℃ for 14h with shaking, and the enzyme was inactivated by boiling in a water bath for 10min. After cooling to 25℃, the mixture was centrifuged at 6500rpm for 15min and the supernatant was collected. The supernatant was precipitated with 50% ethanol, and the supernatant was then precipitated again with 70% ethanol. The supernatant was then further precipitated with 90% ethanol. The final precipitate was reconstituted with distilled water and the residual ethanol was evaporated by rotary evaporation at 45℃. The mixture was then freeze-dried to obtain rice bran ferulic acid oligosaccharide, which was named 90%RBFOs.

[0036] Example 3 A rice bran ferulic acid oligosaccharide

[0037] (1) 250g of fresh rice bran and 550mL of n-hexane were stirred and mixed at 180rpm for 13h to degrease the rice bran. The degreasing process was repeated twice to obtain degreased rice bran. (2) Mix 50g of defatted rice bran with 450mL of distilled water, adjust the pH to 6.5, gelatinize in a 90℃ water bath for 12min, add 4% (w / w) of heat-resistant α-amylase of defatted rice bran for starch hydrolysis for 35min; then adjust the pH to 9, add 2% (w / w) of alkaline protease of defatted rice bran, stir in a 45℃ water bath for 2.5h to remove protein; then adjust the pH to 4, add 2% (w / w) of amyloglucosidase of defatted rice bran, stir in a 50℃ water bath for 0.5h to further degrade residual starch, inactivate enzyme in a boiling water bath for 10min; after cooling to 25℃, centrifuge at 6000rpm for 15min to collect the precipitate, wash the precipitate with deionized water until the supernatant is free of white flocculent matter; dry the washed precipitate at 50℃ for 24h to obtain rice bran insoluble dietary fiber; (3) After the rice bran insoluble dietary fiber was pulverized and passed through an 80-mesh sieve, xylanase solution (xylanase and acetate buffer were mixed at a mass-volume ratio of 100g:1000mL) was added. The mixture was then hydrolyzed in a constant temperature water bath at 60℃ for 13h with shaking, and the enzyme was inactivated in a boiling water bath for 10min. After cooling to 25℃, the mixture was centrifuged at 5500rpm for 15min and the supernatant was collected. The supernatant was precipitated with 50% ethanol, and the supernatant was then precipitated again with 70% ethanol. The supernatant was then further precipitated with 90% ethanol. The final precipitate was reconstituted with distilled water and the residual ethanol was evaporated by rotary evaporation at 45℃. The mixture was then freeze-dried to obtain rice bran ferulic oligosaccharide, which was named 90%RBFOs.

[0038] Comparative Example 1

[0039] Referring to Example 1, the difference from Example 1 is that in step (3) alcohol precipitation, only 50% ethanol is used for precipitation. After evaporating the ethanol from the precipitate, it is freeze-dried to obtain the rice bran ferulic acid oligosaccharide component of this comparative example, named 50%RBFOs. Comparative Example 2 Referring to Example 1, the difference from Example 1 is that in step (3) alcohol precipitation, 50% ethanol is used for precipitation, and the supernatant is precipitated again with 70% ethanol. After the ethanol is evaporated from the precipitate, it is freeze-dried to obtain the rice bran ferulic oligosaccharide component of this comparative example, which is named 70%RBFOs.

[0040] Experimental Example 1: Determination of Chemical Composition

[0041] The neutral sugar, protein, uronic acid, ash and moisture content of 50% RBFOs, 70% RBFOs and 90% RBFOs obtained from Comparative Examples 1-2 and Example 1 were determined, and the results are shown in Table 1. Neutral sugar content: Place the glucose standard in a 50℃ oven for 12 hours. After drying, accurately weigh 50 mg and add deionized water to a 50 mL volumetric flask. The concentration at this point is 1.0 mg / mL. Shake well and take 5 mL of the solution, then add deionized water to a 50 mL volumetric flask to obtain a 0.1 mg / mL glucose standard solution. Take six 15 mL clean, dry stoppered test tubes and add 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the glucose standard solution respectively using a pipette. Add distilled water to each tube to a final volume of 2.0 mL. Place the tubes in a fume hood and add 1.0 mL of 6% phenol solution, followed by 5.0 mL of concentrated sulfuric acid. Shake quickly after adding the concentrated sulfuric acid, incubate in a boiling water bath for 20 minutes, and then cool naturally for 30 minutes. Use a test tube without glucose as a blank control and measure its absorbance at 490 nm. Perform three replicates for each group and take the average value. A standard curve was plotted with glucose concentration (μg / mL) on the x-axis and absorbance on the y-axis. 50 mg of 50% RBFOs, 70% RBFOs, and 90% RBFOs were respectively added to 50 mL volumetric flasks, resulting in a concentration of 1.0 mg / mL. After shaking well, 5 mL of each solution was accurately pipetted into a 50 mL volumetric flask to obtain a 0.1 mg / mL test solution. 1.0 mL of the sample solution was transferred to a 15 mL stoppered test tube, 1.0 mL of distilled water was added, followed by 1.0 mL of 6% phenol solution and 5.0 mL of concentrated sulfuric acid. After adding the concentrated sulfuric acid, the solution was quickly shaken well and allowed to cool naturally. Finally, the measured absorbance values ​​were substituted into the standard curve to obtain the neutral sugar content. The average value was calculated in triplicate. Protein content: Weigh 25 mg bovine serum albumin into a 25 mL volumetric flask, dissolve in a small amount of distilled water, and dilute to volume to obtain a 1.0 mg / mL standard protein solution; take 2.5 mL of the standard protein solution into a 25 mL volumetric flask and dilute to volume to obtain a 0.1 mg / mL protein standard solution; take 6 test tubes and add 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the protein standard solution sequentially, add distilled water to make up to a final volume of 2.0 mL, then add 5.0 mL of Coomassie Brilliant Blue G-250 solution, shake well, and let stand for 5 min. Use the tube without protein standard solution as a blank, and measure the absorbance at 595 nm within 5-20 min; protein concentration (μg / mL) is used as the x-axis, and absorbance is used as the y-axis; take 50 mg of sample and place it in a 50 mL volumetric flask, at which point the concentration is 1.0 mg / mL. After shaking well, accurately pipette 5 mL into a 50 mL volumetric flask to obtain a 0.1 mg / mL test solution. Take 1.0 mL of the sample solution into a 15 mL stoppered test tube, add 1.0 mL of distilled water, and then add 5.0 mL of Coomassie Brilliant Blue G-250 solution. Shake well and let stand for 5 min. Measure the absorbance within 5-20 min. Finally, substitute the measured absorbance value into the protein content in the standard curve, and take the average value in three parallel trials. Glucuronic acid content: Place the galacturonic acid standard in an oven at 50℃ for 10 hours. After removing it, accurately weigh 10 mg and add deionized water to a 10 mL volumetric flask. Shake well and take 1.0 mL of the solution. Add distilled water to a 10 mL volumetric flask to obtain a 0.1 mg / mL galacturonic acid standard solution. Pipette 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the standard solution into stoppered test tubes. Add deionized water to a final volume of 1.0 mL. Add 5 mL of sodium tetraborate-sulfuric acid solution to each tube in an ice-water bath. Remove the tubes and immediately cool to room temperature. Add 0.2 mL of 0.15% carbazole solution to each tube, shake well, and keep at room temperature for 2 hours. Measure the absorbance at 523 nm. Take 50 mg of the sample into a 50 mL volumetric flask. The concentration at this point is 1.0 mg / mL. After shaking well, accurately pipette 5 mL into a 50 mL volumetric flask to obtain a 0.1 mg / mL test solution. Take 1.0 mL of the sample solution into a 15 mL stoppered test tube, add 5 mL of sodium tetraborate-sulfuric acid solution to each tube in an ice-water bath, remove and immediately cool to room temperature, add 0.2 mL of 0.15% carbazole solution to each tube, shake well, keep at room temperature for 2 h, and measure the absorbance at 523 nm. Finally, substitute the measured absorbance value into the standard curve to obtain the uronic acid content. Take the average value of three parallel tests. Ash content: determined according to Method I in GB 5009.4-2016 "National Food Safety Standard - Determination of Ash in Food"; Moisture content: determined according to Method I in GB 5009.3-2016 "National Food Safety Standard - Determination of Moisture in Food"; Table 1 Chemical composition of rice bran ferulic acid oligosaccharides

[0042] The results showed that the neutral sugar content increased significantly with increasing ethanol concentration, reaching the highest level of 84.87 ± 0.85% in the 90% RBFOs component. Simultaneously, the moisture and ash contents gradually decreased with increasing purity, indicating that this component had high purity and few impurities. Regarding other components, the uronic acid content remained relatively high in the 50% and 90% RBFOs components, while the 70% RBFOs component had the lowest uronic acid content and the highest protein content. Therefore, the rice bran ferulic acid oligosaccharide described in Example 1 has higher purity and has the potential to be used as a high-purity oligosaccharide raw material.

[0043] Experimental Example 2: Determination of Ferulic Acid Content

[0044] Ferulic acid standard curve preparation: Accurately weigh the reference standard and add anhydrous ethanol to prepare ferulic acid standard solutions of 25, 50, 75, 100, and 125 μg / mL, respectively. The solutions were then determined by high-performance liquid chromatography (HPLC). Chromatographic conditions: Agilent Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm); column temperature 25℃; data acquisition at 320 nm for 90 min; mobile phase: 1% acetic acid solution (A): methanol (B) = 60:40; flow rate 0.8 mL / min; column temperature 25℃; injection 10 μL. The linear regression equation of peak area y versus mass concentration x was obtained, which is the ferulic acid standard curve. Determination of free ferulic acid content: Weigh 10 mg of 50%RBFOs, 70%RBFOs, and 90%RBFOs powder and dissolve them in 1 mL of methanol solution, then wash 3 times; use high performance liquid chromatography to determine the free ferulic acid content in the 50%RBFOs, 70%RBFOs, and 90%RBFOs samples according to the above chromatographic conditions. Determination of total ferulic acid content: 10 mg of 50% RBFOs, 70% RBFOs, and 90% RBFOs powder were weighed and dissolved in 10 mL of 2 mol / L sodium hydroxide solution and allowed to stand at room temperature for 24 h for complete saponification. The pH was adjusted to 2.0 with 2 mol / L hydrochloric acid solution, and the mixture was extracted three times with 5 mL of ethyl acetate solution. The ethyl acetate extracts were combined and dried completely at 40 °C. The residues were dissolved three times with 1 mL of methanol solution, and the methanol solutions were combined and diluted to a volumetric flask with methanol. The mixture was filtered through a 0.22 μm filter membrane and set aside for use. The total ferulic acid content in 50% RBFOs, 70% RBFOs, and 90% RBFOs was determined by high performance liquid chromatography (HPLC) under the above chromatographic conditions. Bound ferulic acid content: Bound ferulic acid content = Total ferulic acid content - Free ferulic acid content; The results showed that the 50%, 70%, and 90% RBFOs obtained from Comparative Examples 1-2 and Example 1 did not contain free ferulic acid, and the bound ferulic acid contents were 1.51±0.11 μg / g, 10.05±0.14 μg / g, and 19.64±0.17 μg / g, respectively. The bound ferulic acid content was the highest in the 90% RBFOs.

[0045] Experimental Example 3: Molecular Weight Determination

[0046] Preparation of standard solution: Accurately weigh 5 mg of each standard (maltose-P800), dissolve in 1 mL of mobile phase solution to prepare a 5 mg / mL solution, and transfer the sample to a vial; Sample solution preparation: Weigh 5 mg of 50% RBFOs, 70% RBFOs, and 90% RBFOs powder respectively, and dissolve them in 1 mL of mobile phase. Gently shake the centrifuge tube to dissolve the powder, and centrifuge at 12000 rpm for 10 min. Collect the supernatant and filter it through a 0.22 μm aqueous microporous membrane. Then transfer the sample to a vial for chromatographic analysis. The results are shown below. Figure 1 As shown in Table 2; The chromatographic conditions were as follows: mobile phase: 0.2 mol / L sodium chloride solution; column: BRT105-103-101 tandem gel column (8 mm × 300 mm); flow rate: 0.7 mL / min; column temperature: 40 ℃; injection volume: 50 μL; detector: differential detector RID-20A. Table 2. Molecular weight of rice bran ferulic acid oligosaccharides

[0047] The results showed that the molecular weight of the three feruloyl oligosaccharides, 50% RBFOs, 70% RBFOs and 90% RBFOs, decreased sequentially, and the molecular weight uniformity was close to 1, indicating that the molecular weight was uniform.

[0048] Experiment Example 4: Determination of Monosaccharide Composition

[0049] Preparation of standard solutions: Prepare a standard stock solution by taking 8 monosaccharide standards (rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, and glucuronic acid); Sample solution preparation: Accurately weigh 5 mg of 50% RBFOs, 70% RBFOs, and 90% RBFOs powder and place them in an ampoule. Add 2 mL of 3 mol / L trifluoroacetic acid and hydrolyze at 120℃ for 3 h. Accurately pipette the acid-hydrolyzed solution into a tube and dry it under nitrogen. Add 5 mL of water and vortex to mix. Pipe 200 µL of 50% RBFOs into 800 µL of deionized water, pipette 50 µL of 70% RBFOs into 950 µL of deionized water, and pipette 100 µL of 90% RBFOs into 900 µL of deionized water. Centrifuge at 12000 rpm for 5 min. Filter the supernatant through a 0.22 µm microporous membrane and perform chromatographic analysis. The results are as follows: Figure 2 As shown in Table 3; Chromatographic conditions: Column: Dionex Carbopac™ PA20 (3mm × 150mm); Mobile phase: A: H2O; B: 15mM NaOH; C: 15mM NaOH & 100mM NaOAc; Flow rate: 0.3mL / min; Injection volume: 25µL; Column temperature: 30℃; Elution gradient as follows: 0min A / B / C (99:1:0, V / V), 18min A / B / C (99:1:0, V / V), 20min A / B / C (75:25:0, V / V), 30min A / B / C (75:25:0, V / V), 30.1min A / B / C (20:0:80, V / V), 46min A / B / C (20:0:80, V / V), 46.1min Phase A / Phase B / Phase C (0:100:0, V / V), 50 min Phase A / Phase B / Phase C (0:100:0, V / V), 50.1 min Phase A / Phase B / Phase C (99:1:0, V / V), 80 min Phase A / Phase B / Phase C (99:1:0, V / V); Detector: Electrochemical detector; Table 3 Monosaccharide composition of rice bran ferulic acid oligosaccharides

[0050] The results showed that 50% RBFOs, 70% RBFOs, and 90% RBFOs were mainly composed of monosaccharides such as arabinose, xylose, galactose, and mannose, but the proportions of each component varied significantly among different samples. The glucose content was low and varied in all three types of RBFOs, indicating that there was little residual starch. In addition, the molar ratios (A / X) of arabinose to xylose were 1.09, 0.91, and 1.13, respectively, with the A / X ratios close to 1, suggesting that all three types of RBFOs had a high degree of side chain substitution.

[0051] Experimental Example 5: Ultraviolet Spectroscopy Measurement

[0052] 50%, 70%, and 90% RBFOs were dissolved in PBS buffer to prepare a 0.25 mg / mL solution. UV spectroscopy was performed in the 200–400 nm range, and the results are as follows: Figure 3 As shown; The results showed that 50%RBFOs, 70%RBFOs and 90%RBFOs all had obvious absorption peaks around 206 nm, with the 90%RBFOs showing the strongest absorption peak, indicating that the three oligosaccharides had high sugar content, with the 90%RBFOs having the highest sugar content. In addition, an obvious absorption peak was also observed at 326 nm, indicating that all three oligosaccharides contained ferulic acid.

[0053] Experimental Example 6: Fourier Transform Infrared Measurement

[0054] 50% RBFOs, 70% RBFOs, and 90% RBFOs powders were respectively mixed with potassium bromide powder at a mass ratio of 1:100, pressed into discs, and then pressed at a pressure of 400~4000 cm⁻¹. -1 The spectrum was recorded within the frequency range, and the sample was scanned 32 times; the results are as follows. Figure 4 As shown; The results showed that in the infrared spectra of 50% RBFOs, 70% RBFOs, and 90% RBFOs, 1541.33 cm⁻¹... -1 1550.49cm -1 and 1523.01cm -1 The absorption peak at 1730.80 cm⁻¹ is caused by the stretching vibration of C=C in the aromatic ring, indicating that the sample contains ferulic acid. -1 1728.87cm -1 and 1725.98cm -1 The absorption peak at the point is caused by the C=O of the carbonyl group, indicating that ferulic acid is linked to the oligosaccharide through an ester bond; and a red shift occurred, with the degree of red shift increasing sequentially, indicating that the interaction between the sugar chain and ferulic acid is the strongest in 90% RBFOs.

[0055] Experimental Example 7: Effect of rice bran feruloyl oligosaccharides on α-amylase activity

[0056] Three types of rice bran feruloyl oligosaccharides (50% RBFOs, 70% RBFOs, 90% RBFOs), acarbose, α-amylase, and rice starch solutions were prepared using 0.1 mol / L pH 6.8 phosphate buffer. The concentrations of 50% RBFOs, 70% RBFOs, and 90% RBFOs were 10, 30, 50, 100, and 200 µg / mL, respectively, with acarbose serving as a positive control. 200 µL of rice bran feruloyl oligosaccharide and acarbose solutions of different concentrations were added to each test tube, followed by 200 µL of 10 U / mL α-amylase solution. The mixtures were thoroughly mixed and incubated at 37°C for 15 min. Then, 200 µL of fully gelatinized 1% (w / w) rice starch solution was added and incubated at 37°C for 20 min. Finally, 300 µL of... DNS reagent was used for color development, and the reaction was immediately terminated by boiling in a water bath for 5 minutes. After the reaction system was immediately cooled to room temperature, 50 mL of distilled water was added for dilution and mixed well. The absorbance of sample A was measured at 540 nm. An equal volume of PBS solution was used instead of the α-amylase solution to measure the absorbance of control A. In the blank group, an equal volume of PBS solution was used instead of the sample solution to measure the absorbance of blank A. The α-amylase inhibition rate was calculated according to the formula, and the results are as follows: Figure 5 As shown; Inhibition rate (%) = [1 - (Sample A - Control A) / Blank A] × 100; The results showed that the inhibition rates of the three oligosaccharides (50% RBFOs, 70% RBFOs, and 90% RBFOs) against α-amylase increased with increasing concentration, exhibiting a typical dose-response relationship. Specifically, when the concentration of the three oligosaccharides increased from 10 µg / mL to 200 µg / mL, their inhibition rates increased from 11.29±0.44%, 15.20±0.25%, 15.49±0.39%, and 21.24±0.18% to 33.16±0.40%, 41.36±0.37%, and 4%, respectively. The half-maximal inhibitory concentrations (IC50) of 50%, 70%, and 90% RBFOs against α-amylase activity were 344.36±4.04 µg / mL, 268.62±4.67 µg / mL, and 234.39±4.48 µg / mL, respectively, all higher than that of acarbose (111.32±1.81 µg / mL), but the differences were not significant. 90% RBFOs showed better inhibitory effects on α-amylase than acarbose.

[0057] Experimental Example 8: Effect of rice bran feruloyl oligosaccharides on α-glucosidase activity

[0058] Three rice bran ferulic acid oligosaccharide components, acarbose, α-glucosidase, and pNPG solutions were prepared using 0.1 mol / L phosphate buffer solution (pH 6.8). The concentrations of 50%, 70%, and 90% RBFOs were 10, 30, 50, 100, and 200 µg / mL, respectively, with acarbose serving as a positive control. 30 µL of each concentration of sample and acarbose solution were added to each well of a 96-well plate, followed by 30 µL of 3 U / mL α-glucosidase solution. The plates were incubated at 37°C for 15 min, then 30 µL of 5 mmol / L pNPG solution was added, and the plates were incubated at 37°C for 20 min. Finally, 200 µL of pNPG solution was added to each well. The reaction was terminated with 1 mol / L sodium carbonate solution, and the absorbance of sample A was measured at 405 nm. In the sample control group, 30 µL of PBS solution was used instead of the enzyme solution in the sample group, and the absorbance of control A was measured. In the blank group, 30 µL of PBS solution was used instead of the sample solution, and the absorbance of blank A was measured. The α-glucosidase inhibition rate was calculated according to the formula, and the results are as follows. Figure 6 As shown; Inhibition rate (%) = [1 - (Sample A - Control A) / Blank A] × 100; The results showed that the inhibitory activities of 50% RBFOs, 70% RBFOs, 90% RBFOs, and acarbose on α-glucosidase were dose-dependent. At 200 µg / mL, the inhibition rates of 50% RBFOs, 70% RBFOs, 90% RBFOs, and acarbose on α-glucosidase were 50.17 ± 0.58%, 98.06 ± 0.04%, 98.58 ± 0.10%, and 99.81 ± 0.02%, respectively. The IC50 of the samples was analyzed. 50 Comparison of values, IC50 of acarbose 50 The lowest value indicates the strongest inhibitory ability, followed by 90% RBFOs; this suggests that 90% RBFOs have better enzyme inhibitory activity compared to 50% RBFOs and 70% RBFOs.

[0059] Experimental Example 9: Kinetic Determination of the Inhibition of α-Amylase and α-Glucosidase Activities by Rice Bran Feruloyl Oligosaccharides

[0060] With the α-amylase concentration fixed at 10 U / mL, the concentrations of three types of rice bran feruloyl oligosaccharides (50% RBFOs, 70% RBFOs, and 90% RBFOs) were varied to 10, 20, 50, 100, and 200 µg / mL, and the rice starch concentration was varied to 0.05, 1, 2, and 3 mg / mL. The enzyme reaction rate (v), substrate concentration (S), and inhibitor concentration (I) were measured. The inhibition type of α-amylase by the 50% RBFOs, 70% RBFOs, and 90% RBFOs obtained in proportions 1-2 and Example 1 was determined using the Lineweaver-Burk equation. The results are as follows: Figure 7 As shown; With the α-glucosidase concentration fixed at 3 U / ml, the concentrations of three rice bran ferulic acid oligosaccharide components were varied to 10, 30, 50, 100, and 200 µg / mL, and the substrate pNPG solution concentration was varied to 1, 3, 5, and 7 mmol / L. The enzyme reaction rate v, substrate concentration [S], and inhibitor concentration [I] were measured. The inhibition type of α-glucosidase by 50% RBFOs, 70% RBFOs, and 90% RBFOs obtained in proportions 1-2 and Example 1 was determined using the Lineweaver-Burk equation. The results are as follows: Figure 7 As shown; The results showed that the Lineweaver-Burk curves of the three rice bran feruloyl oligosaccharides against α-amylase and α-glucosidase all intersected in the second quadrant. Furthermore, the slope of the curves increased with increasing inhibitor concentration, accompanied by an increase in Km value and a decrease in Vmax value. This kinetic behavior indicates that the inhibition mode is mixed inhibition, and its mode of action is homologous to acarbose. 50%, 70%, and 90% RBFOs partially bound to the enzyme's active site, exhibiting competitive inhibition; others bound to the enzyme surface, exhibiting non-competitive inhibition, meaning they not only competed with the enzyme for substrate but also bound to the enzyme-starch complex. Therefore, the three rice bran feruloyl oligosaccharide components mainly affect enzyme activity by altering the enzyme's conformation and microenvironment.

[0061] As can be seen from the above embodiments, the present invention provides a rice bran feruloyl oligosaccharide, its preparation method and application. The prepared rice bran feruloyl oligosaccharide can significantly inhibit the activity of α-amylase and α-glucosidase, and can be used as a food-derived hypoglycemic component for the development of functional foods, medicines and other products for the purpose of regulating blood sugar. It provides a new material basis for the development of novel, low-toxicity natural inhibitors of postprandial hyperglycemia.

[0062] 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 rice bran ferulic acid oligosaccharides, characterized in that, Includes the following steps: (1) Mix rice bran and n-hexane to defatt the rice, and obtain defatted rice bran; (2) The defatted rice bran was gelatinized and enzymatically hydrolyzed to obtain insoluble dietary fiber from rice bran; (3) Enzymatically hydrolyze and centrifuge the insoluble dietary fiber of rice bran, and take the supernatant; use 50% ethanol, 70% ethanol and 90% ethanol to perform gradient alcohol precipitation of the supernatant to obtain rice bran feruloyl oligosaccharide.

2. The preparation method according to claim 1, characterized in that, In step (1), the mass-to-volume ratio of the rice bran and n-hexane is 250 mg: 450~550 mL; the degreasing time is 12~14 h; stirring is required during the degreasing process, and the stirring speed is 160~180 rpm; the degreasing is performed 1~3 times.

3. The preparation method according to claim 1, characterized in that, In step (2), the gelatinization step is as follows: mix defatted rice bran and water, adjust the pH to 5.5~6.5, and gelatinize at 90~100℃ for 8~12 minutes to obtain gelatinized defatted rice bran; The mass-to-volume ratio of the defatted rice bran and water is 50 mg: 450~550 mL.

4. The preparation method according to claim 3, characterized in that, In step (2), the enzymatic hydrolysis includes the following steps: the gelatinized defatted rice bran is mixed with heat-resistant α-amylase, alkaline protease and amyloglucosidase for enzymatic hydrolysis.

5. The preparation method according to claim 4, characterized in that, The amount of the heat-resistant α-amylase added is 4% of the mass of defatted rice bran; the enzymatic hydrolysis conditions of the defatted rice bran and the heat-resistant α-amylase are: enzymatic hydrolysis at 90~100℃ for 25~35 min; The amount of alkaline protease added is 2% of the mass of defatted rice bran; the enzymatic hydrolysis conditions of the alkaline protease are: adjust the pH to 8-9, and hydrolyze at 45-55℃ for 1.5-2.5 hours. The amount of amyloglucosidase added is 2% of the mass of defatted rice bran; the enzymatic hydrolysis conditions of the amyloglucosidase are: adjust the pH to 4-5, and hydrolyze at 50-60℃ for 0.5-1.5h.

6. The preparation method according to claim 1, characterized in that, In step (3), the enzymatic hydrolysis step is as follows: the rice bran insoluble dietary fiber and xylanase solution are enzymatically hydrolyzed at 50~60℃ for 12~14h; The mass-to-volume ratio of the rice bran insoluble dietary fiber to the xylanase solution is 100 mg: 1000 mL.

7. Rice bran feruloyl oligosaccharide is prepared by the preparation method according to any one of claims 1 to 6.

8. The use of rice bran feruloyl oligosaccharide prepared by the preparation method according to any one of claims 1 to 6 or the rice bran feruloyl oligosaccharide according to claim 7 in the preparation of products that inhibit the activity of α-amylase and α-glucosidase.

9. The rice bran feruloyl oligosaccharide prepared by the preparation method according to any one of claims 1 to 6, or the rice bran feruloyl oligosaccharide according to claim 7, is used in the preparation of products for regulating blood sugar.

10. The rice bran feruloyl oligosaccharide prepared by the preparation method according to any one of claims 1 to 6, or the rice bran feruloyl oligosaccharide according to claim 7, is used in the preparation of products for treating diabetes.

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