A starch-quercetin complex enzymatic hydrolysate, its preparation method and application

Abstract

This invention discloses a starch-quercetin complex enzymatic hydrolysate, its preparation method, and its application, relating to the field of starch preparation technology. The preparation method of the starch-quercetin complex enzymatic hydrolysate includes the following steps: mixing starch and water, heating and gelatinizing to obtain a starch paste; mixing the starch paste and quercetin, stirring to obtain a starch-quercetin complex gel; and mixing the starch-quercetin complex gel and pullulanase under pullulanase enzymatic hydrolysis temperature and pH conditions to perform enzymatic debranching reaction, obtaining the starch-quercetin complex enzymatic hydrolysate. By mixing starch and quercetin to form a complex before enzymatic debranching treatment, the local inhibitory effect of quercetin on the starch debranching process is utilized, achieving directional regulation of starch chain length distribution, selectively enriching the long branches in DP25-76, ultimately reducing the content of rapidly digestible starch and increasing the content of slowly digestible starch and resistant starch, thus altering the digestibility of starch.

Description

Technical Field

[0001] This invention relates to the field of starch preparation technology, and in particular to a starch-quercetin complex enzymatic hydrolysate, its preparation method, and its application. Background Technology

[0002] Natural starch consists of alternating ordered and amorphous regions of a double helix, exhibiting a semi-crystalline structure and protected by a dense granular shell. This makes it difficult for α-amylase to directly access the internal glycosidic bonds. Therefore, ungelatinized natural starch is almost entirely undigested in the human small intestine, resulting in a generally low content of rapidly digestible starch. However, once natural starch is heated and gelatinized, the granular structure breaks down, the double helix unwinds, and enzymes can act freely, leading to a dramatic increase in the content of rapidly digestible starch. Furthermore, this rapidly digestible starch is quickly broken down into glucose after ingestion, causing a sharp rise in postprandial blood sugar and stimulating a large secretion of insulin, which is detrimental to human health.

[0003] To reduce the digestibility of starch, physical, chemical, or enzymatic modifications are commonly used. Enzymatic modification, also known as enzymatic debranching, involves enzymes such as Prussian blue enzymes specifically cleaving the α-1,6 glycosidic bonds in starch molecules to generate linear short chains. These linear short chains can form a more ordered crystalline structure during retrogradation, thereby increasing the content of slow-digesting starch and resistant starch.

[0004] In starch branched chains, short branches are generally considered to be the starting sites for amylase activity because they are difficult to form ordered helices. However, traditional enzymatic debranching methods suffer from poor selectivity and imprecise control over starch chain length distribution, and the starch hydrolysates obtained by traditional enzymatic debranching methods have a high content of rapidly digestible starch. Summary of the Invention

[0005] The main objective of this invention is to propose a starch-quercetin complex enzymatic hydrolysate, its preparation method, and its application, aiming to solve the problem that the starch hydrolysates obtained by enzymatic debranching in the prior art have a high content of rapidly digestible starch.

[0006] To achieve the above objectives, this invention proposes a method for preparing a starch-quercetin complex enzymatic hydrolysate, the method comprising the following steps:

[0007] S1. Mix starch and water, heat to gelatinize, and obtain starch paste; S2. Mix the starch paste and quercetin, stir, and obtain a starch-quercetin composite gel; S3. Under the enzymatic hydrolysis temperature and pH conditions of pullulanase, the starch-quercetin composite gel and pullulanase are mixed and subjected to enzymatic debranching reaction to obtain starch-quercetin composite hydrolysate.

[0008] In one embodiment, in step S1: The temperature for heating and gelatinizing is 85-95℃; and / or, The heating and gelatinization time is 10-20 minutes.

[0009] In one embodiment, in step S2: The mass ratio of quercetin to starch is 1-5:100; and / or, The stirring time is 1-2 hours.

[0010] In one embodiment, in step S3: The enzymatic hydrolysis temperature is 45-55℃; and / or, The enzymatic hydrolysis pH is 4.5-5.5; and / or, Add 10-30 ASPU pullulanase per 1g of starch; and / or, The enzymatic debranching reaction takes 5-90 minutes.

[0011] In one embodiment, step S3 includes: Under the enzymatic hydrolysis temperature and pH conditions of pullulanase, starch-quercetin composite gel and pullulanase were mixed and subjected to enzymatic debranching reaction. The precipitate was collected, washed with anhydrous ethanol, centrifuged, and freeze-dried to obtain starch-quercetin composite hydrolysate.

[0012] This invention also provides a starch-quercetin complex enzymatic hydrolysate, which is prepared by the aforementioned method for preparing starch-quercetin complex enzymatic hydrolysates; the starch-quercetin complex enzymatic hydrolysate comprises a starch derivative and quercetin bound to the starch derivative via a V-shaped single-helix inclusion structure: The branched chain length distribution of the starch derivative is as follows: the proportion of branched segments with a degree of polymerization of DP25-36 is ≥16%, and the proportion of branched segments with a degree of polymerization of DP37-76 is ≥20%. In the starch-quercetin complex enzymatic hydrolysate: the mass percentage of rapidly digestible starch is less than 55%, and the mass percentage of resistant starch is greater than 30%.

[0013] The present invention also provides the application of starch-quercetin complex enzymatic hydrolysate in low glycemic index foods, slow-release energy foods or functional health foods, wherein the starch-quercetin complex enzymatic hydrolysate includes the starch-quercetin complex enzymatic hydrolysate prepared by the aforementioned method or the aforementioned starch-quercetin complex enzymatic hydrolysate.

[0014] In the technical solution of this invention, starch and quercetin are first mixed to form a complex, and then enzymatic debranching is performed. This utilizes the local inhibition of starch debranching by quercetin, achieving directional regulation of starch chain length distribution. It selectively enriches the long branches in DP25-76, ultimately reducing the content of rapidly digestible starch and increasing the content of slowly digestible starch and resistant starch, fundamentally changing the digestibility of starch and yielding a slow-release carbohydrate. Furthermore, this complex enzymatic hydrolysate has a more stable V-shaped crystal structure and a higher gelatinization enthalpy, significantly improving thermal stability. The entire preparation process uses food-grade enzymes and natural polyphenols, requiring no chemical reagents, making it safe, environmentally friendly, and in line with the development trend of clean-label foods. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0016] Figure 1 The diagram shows the branch chain length distribution of gelatinized starch, starch-quercetin complex, starch-quercetin complex enzymatic hydrolysates prepared in Examples 1-4, and starch enzymatic hydrolysates prepared in Comparative Examples 1-4. Figure 2(A) shows the infrared spectra of the starch-quercetin complex enzymatic hydrolysate and quercetin prepared in Examples 1-5 of the present invention; Figure 2(B) shows the infrared spectra of the starch enzymatic hydrolysate and natural starch prepared in Comparative Examples 1-5 of the present invention; Figure 2(C) shows the infrared spectra of gelatinized starch, starch-quercetin complex, starch-quercetin complex enzymatic hydrolysate prepared in Examples 1-5, and starch enzymatic hydrolysate prepared in Comparative Examples 1-5 at 1047 cm⁻¹. -1 Infrared intensity at 1022 cm -1 The result of the infrared ratio of the infrared intensity at the location; Figure 3(A) shows the TGA test results of gelatinized starch, starch-quercetin complex, starch-quercetin complex enzymatic hydrolysate prepared in Examples 1-5, and starch enzymatic hydrolysate prepared in Comparative Examples 1-5 in this invention; Figure 3(B) shows the DGT results of gelatinized starch, starch-quercetin complex, starch-quercetin complex enzymatic hydrolysate prepared in Examples 1-5, and starch enzymatic hydrolysate prepared in Comparative Examples 1-5 in this invention. Figure 4(A) shows the DSC curves of the starch hydrolysate, gelatinized starch and natural starch prepared in Comparative Examples 1-5 of this invention; Figure 4(B) shows the DSC curves of the starch-quercetin complex in this invention and the starch-quercetin complex hydrolysate prepared in Examples 1-5. Figure 5(A) shows the XRD patterns of natural starch and quercetin in this invention; Figure 5(B) shows the XRD patterns of the starch-quercetin complex in this invention and the starch-quercetin complex enzymatic hydrolysates prepared in Examples 1-5; Figure 5(C) shows the XRD patterns of gelatinized starch and the starch enzymatic hydrolysates in Comparative Examples 1-5. Figure 6 shows the XPS analysis chromatograms of natural starch, starch-quercetin complex hydrolysates prepared in Examples 3-4, starch-quercetin complex, and starch hydrolysates prepared in Comparative Examples 3-4 in this invention; Figure 6(A) is the full spectrum, Figure 6(B) is the percentage graph of C and O content; Figure 6(C) is the high-resolution spectrum of C; Figure 6(D) is the high-resolution spectrum of O. Figure 7 This is a scanning electron microscope (SEM) image of quercetin in this invention; Figure 8 The images show SEM images (magnification 1000) of the gelatinized starch, starch-quercetin complex, starch hydrolysates prepared in Comparative Examples 1-5, and starch-quercetin complex hydrolysates prepared in Examples 1-5. Figure 9 The images show SEM images (magnification 2000) of the gelatinized starch, starch-quercetin complex, starch hydrolysates prepared in Comparative Examples 1-5, and starch-quercetin complex hydrolysates prepared in Examples 1-5. Figure 10 This is a graph showing the degree of complexation of starch-quercetin complexes with different quercetin contents in this invention.

[0017] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, or solution B, or a solution where both A and B are satisfied simultaneously. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Traditional enzymatic debranching has problems such as poor selectivity and insufficient precision in controlling the distribution of starch chain length. Furthermore, the starch hydrolysate obtained by traditional enzymatic debranching has a high content of rapidly digestible starch.

[0020] In view of this, the present invention proposes a method for preparing a starch-quercetin complex enzymatic hydrolysate, the method comprising the following steps: S1, mixing starch and water, heating and gelatinizing to obtain a starch paste; S2, mixing the starch paste and quercetin, stirring to obtain a starch-quercetin complex gel; S3, under the enzymatic hydrolysis temperature and pH conditions of pullulanase, mixing the starch-quercetin complex gel and pullulanase to carry out an enzymatic debranching reaction to obtain the starch-quercetin complex enzymatic hydrolysate.

[0021] In the technical solution of this invention, starch and quercetin are first mixed to form a complex, and then enzymatic debranching is performed. This utilizes the local inhibition of starch debranching by quercetin, achieving directional regulation of starch chain length distribution. It selectively enriches the long branches in DP25-76, ultimately reducing the content of rapidly digestible starch and increasing the content of slowly digestible starch and resistant starch, fundamentally altering the digestibility of starch and yielding a slow-release carbohydrate, namely, a starch-quercetin complex enzymatic hydrolysate. Furthermore, this complex enzymatic hydrolysate has a more stable V-shaped crystal structure and a higher gelatinization enthalpy, significantly improving thermal stability. The entire preparation process uses food-grade enzymes and natural polyphenols, requiring no chemical reagents, making it safe, environmentally friendly, and in line with the development trend of clean-label foods.

[0022] In step S1, the starch is preferably cereal starch, which can be starch from low-cadmium rice.

[0023] Among them, rapidly digestible starch (RDS) is rapidly enzymatically broken down and absorbed in the small intestine, usually within 20 minutes; slowly digestible starch (SDS) is slowly and continuously enzymatically broken down and absorbed in the small intestine, providing a stable release of energy and correspondingly stable blood glucose levels, with a hydrolysis time of 20-120 minutes; resistant starch (RS) is not or is minimally broken down and absorbed by enzymes in the small intestine, but can reach the large intestine, where it is fermented and utilized by intestinal microorganisms as a prebiotic.

[0024] It should be noted that in the technical solution of this invention, starch and quercetin are first mixed to form a complex. Quercetin can inhibit the subsequent degradation of long branched chains in starch by Prussian blue enzyme, thereby ensuring that the final complex hydrolysate has a high proportion of medium and long branched chains and a low proportion of rapidly digestible starch. By utilizing the chain length dependence of the V-shaped single-helix inclusion structure and the debranching effect of debranching enzymes on starch, the distribution of starch chain length can be precisely controlled to a certain extent, and the slow digestibility of the complex can be synergistically improved.

[0025] It should be noted that when the enzymatic hydrolysis time is 30 min, the proportion of medium and long branched chains (i.e., DP25-36 and DP37-76) in the prepared starch-quercetin complex hydrolysate is relatively high, while the proportion of short branched chains (DP6-12) is relatively low. Under these conditions, the complex contains lower levels of rapidly digestible starch and higher levels of slowly digestible starch and resistant starch, increasing the difficulty of digestion and making it less likely to cause a rapid rise in blood sugar after ingestion. This is because quercetin in the product after 30 min of enzymatic hydrolysis has a certain inhibitory effect on Prussian enzymes, which can retain more medium and long chains. These medium and long chains promote the formation of a dense and stable crystal structure and, through the protective effect on branch points and steric hindrance, jointly slow down the erosion rate of digestive enzymes.

[0026] Natural starch undergoes enzymatic debranching after gelatinization to obtain starch hydrolysate. The molecular weight of starch hydrolysate is lower than that of natural starch, and the molecular weight of starch hydrolysate continues to decrease with the extension of enzymatic debranching time. In contrast, the molecular weight of starch-quercetin complex is greater than that of natural starch, and its molecular weight decreases rapidly after enzymatic debranching. This is because the special structure of the complex may cause pullulanase cleavage sites to be very concentrated, tending to produce chains with more uniform length and shorter length, even lower than the molecular weight of starch hydrolysate.

[0027] In infrared testing at 1047 cm -1 and 1022 cm -1 The infrared intensity ratio represents the ratio of the infrared intensity of the ordered crystalline and amorphous regions. After enzymatic debranching, the infrared intensity ratio of natural starch first increases and then decreases to a level similar to that of natural starch. However, the infrared intensity ratio increases after natural starch is combined with quercetin, indicating that the introduction of quercetin induces significant structural ordering of starch molecules. The main reason is that quercetin, as a ligand, promotes the formation of regular V-shaped single-helix inclusion complexes of starch chains, thereby enhancing the short-range ordered structure of the system. As the enzymatic debranching time of the starch-quercetin complex increases, its infrared intensity ratio decreases significantly and is significantly lower than that of starch hydrolysate. This indicates that the starch-quercetin complex hydrolysate has a stronger disorder, mainly because it produces a large number of short-chain, low-molecular-weight oligosaccharides, whose structural disorder is much higher than that of the enzymatic hydrolysate of natural starch.

[0028] It should also be noted that natural starch, after enzymatic debranching, shows no XRD diffraction peaks and no new crystalline regions, exhibiting a loose structure. In contrast, the starch-quercetin complex, after enzymatic debranching, shows multiple XRD diffraction peaks, indicating increased crystallinity. Quercetin is exposed from the interior of the complex to the surface, exerting a hydrophobic effect. Therefore, compared to starch hydrolysates, the starch-quercetin complex hydrolysate provided by this invention has higher crystallinity, resulting in a higher initial decomposition temperature and higher thermal stability. Furthermore, compared to starch hydrolysates, the starch-quercetin complex hydrolysate exhibits increased gelatinization temperature and enthalpy, with the gelatinization temperature gradually increasing to a limit as the debranching time increases. This process demonstrates good structural stability of the complex hydrolysate, indicating that it is less prone to gelatinization during cooking. Natural starch, on the other hand, does not show melting peaks after debranching because the gelatinization process completely disintegrates the starch granules, and the debranching treatment does not form a crystalline structure, unlike the gelatinization tendency of the complex hydrolysate of this invention.

[0029] It should be noted that the product obtained by preparing natural starch into starch paste, then simultaneously adding quercetin and Prussian blue enzyme for enzymatic hydrolysis, collecting the precipitate, and freeze-drying is different from that of this application. Furthermore, this method cannot achieve the effect of a high proportion of medium and long branched chains in the starch-quercetin complex enzymatic hydrolysate of this application. The specific reason is that the starch is not pre-complexed with quercetin, so a specific complex structure cannot be formed, the enzymatic hydrolysis process cannot be controlled, and it is difficult to exert the effect of enzymatic hydrolysis of specific short chains.

[0030] In some embodiments, in step S1: the temperature for heating and gelatinizing is 85-95°C; and / or the time for heating and gelatinizing is 10-20 minutes. That is, the temperature for heating and gelatinizing can be 85°C, 90°C, or 95°C, and the time for heating and gelatinizing can be 10 minutes, 15 minutes, or 20 minutes. Controlling the temperature and time for heating and gelatinizing within the above range can ensure that the starch gelatinization is more thorough.

[0031] In some embodiments, in step S2: the mass ratio of quercetin to starch is 1-5:100; and / or, the stirring time is 1-2 hours. The mass ratio of quercetin to starch can be 1:100, 2:100, 3:100, 4:100, or 5:100, and the stirring time can be 1 hour, 1.5 hours, or 2 hours. Controlling the mass ratio and stirring time within the above ranges ensures that quercetin and starch are fully mixed to form a composite gel, allowing quercetin to be more fully encapsulated within the hydrophobic cavities of the starch, thus enabling it to inhibit the enzymatic hydrolysis of long-branched chains in starch during the subsequent enzymatic debranching step. Preferably, the mass ratio of quercetin to starch is 2.5:100.

[0032] In some embodiments, in step S3: the enzymatic hydrolysis temperature is 45-55℃; and / or, the enzymatic hydrolysis pH is 4.5-5.5; and / or, 10-30 U of pullulanase is added per 1g of starch; and / or, the enzymatic debranching reaction time is 5-90 min. The enzymatic hydrolysis temperature can be 45℃, 50℃, or 55℃; the enzymatic hydrolysis pH can be 4.5, 5.0, or 5.5; the amount of Prussian blue enzyme can be 10 ASPU / g starch, 20 ASPU / g starch, or 30 ASPU / g starch; and the enzymatic debranching reaction time can be 5 min, 20 min, 40 min, 60 min, 80 min, or 90 min. Simultaneously controlling the enzymatic hydrolysis temperature, enzymatic hydrolysis pH, Prussian blue enzyme dosage, and enzymatic debranching reaction time within the above ranges ensures that the enzymatic debranching is performed to a suitable degree, and the medium and long branches of starch are well preserved. Preferably, the enzymatic debranching reaction temperature is 30 min.

[0033] In some embodiments, step S3 includes: mixing the starch-quercetin composite gel and pullulanase under the enzymatic hydrolysis temperature and pH conditions of pullulanase, performing an enzymatic debranching reaction, collecting the precipitate, washing the precipitate with anhydrous ethanol, centrifuging, and freeze-drying to obtain the starch-quercetin composite enzymatic hydrolysate. In this step, washing with anhydrous ethanol also removes free quercetin and terminates the reaction.

[0034] This invention also provides a starch-quercetin complex enzymatic hydrolysate, which is prepared by the aforementioned method for preparing starch-quercetin complex enzymatic hydrolysates. The starch-quercetin complex enzymatic hydrolysate comprises a starch derivative and quercetin bound to the starch derivative via a V-shaped single-helix inclusion structure. The branched chain length distribution of the starch derivative is as follows: the proportion of branched segments with a degree of polymerization (DP) of 25-36 is ≥16%, and the proportion of branched segments with a degree of polymerization (DP) of 37-76 is ≥20%. In the starch-quercetin complex enzymatic hydrolysate, the mass percentage of rapidly digestible starch is less than 55%, and the mass percentage of resistant starch is >30%. Therefore, it possesses all the beneficial effects of the aforementioned starch-quercetin complex enzymatic hydrolysate, which will not be elaborated further here.

[0035] This invention also provides the application of a starch-quercetin complex enzymatic hydrolysate in low glycemic index foods, slow-release energy foods, or functional health foods. The starch-quercetin complex enzymatic hydrolysate includes the starch-quercetin complex enzymatic hydrolysate prepared by the aforementioned method or the aforementioned starch-quercetin complex enzymatic hydrolysate itself. Therefore, it possesses all the beneficial effects of the aforementioned starch-quercetin complex enzymatic hydrolysate or the aforementioned method for preparing it, which will not be elaborated upon here.

[0036] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0037] The experimental indicators and testing methods involved in the embodiments of the present invention are as follows: (1) Determination of chain length of amylopectin Weigh 10 mg of sample, resuspend in 5 mL of water, and incubate in a boiling water bath for 60 min, vortexing intermittently. Add 50 μL of sodium acetate (final concentration 0.6 M, pH 4.4), NaN3 (final concentration 10 μL, 2% w / v), and 10 μL of isoamylase (1400 U), and incubate at 37 °C for 24 h. Add 0.5% (w / v) sodium borohydride solution, vortex, and let stand for 20 h. Transfer 600 μL of the above mixture to a centrifuge tube, dry under nitrogen at room temperature, then dissolve in 30 μL of 1 M NaOH for 60 min, dilute with 570 μL of water, centrifuge at 12000 rpm for 5 min, and load the supernatant onto the sample. The chromatographic system used is a Thermo ICS5000 ion chromatography system (ICS5000+, ThermoFisher Scientific, USA), and starch is analyzed and detected using an electrochemical detector. A Dionex™ CarboPac™ PA200 (250 × 4.0 mm, 10 μm) liquid chromatography column was used, with an injection volume of 5 μL. Mobile phase A: 0.2 M NaOH; Mobile phase B: 0.2 M NaOH / 0.2 M NaAC; Column temperature: 30 °C. Components were analyzed using an electrochemical detector. Flow rate: 0.4 mL / min; Elution gradient: 0 min A / B (90:10 V / V), 10 min A / B (90:10 V / V), 30 min A / B (40:60 V / V), 50 min A / B (40:60 V / V); 50.1 min A / B (90:10 V / V); 60 min A / B (90:10 V / V).

[0038] (2) Determination of molecular weight Weigh 5 mg of sample, add 5 mL of mobile phase DMSO, heat at 80℃ for 3 h to dissolve, filter through a 0.45 μm filter, and then analyze. Use a gel size exclusion column with an appropriate molecular weight range (Ohpak SB-805 HQ (300×8 mm), Ohpak SB-804 HQ (300×8 mm), Ohpak SB-803 HQ (300×8 mm)); column temperature 60℃; injection volume 200 μL; mobile phase A (0.5% LiBr, DMSO); flow rate 0.3 mL / min; elution gradient: isocratic for 120 min; DMSO solution: dn / dc value 0.07 mL / g. Based on the sample quantification results, calculate Mn (number-average molecular weight); Mw (weight-average molecular weight); Mz (z-average molecular weight); Mp (peak molecular weight, the molecular weight of the most abundant component fragment); and Mw / Mn (polydispersity index, the molecular weight distribution width index).

[0039] (3) Measurement of thermal properties Accurately weigh 3.0 mg of sample and place it in a DSC crucible. Add 9 μL of ultrapure water, stir and mix well, seal and store at room temperature for 24 h. The blank control is an empty crucible. Set the heating conditions to 30-120 °C at a heating rate of 10 °C / min. Analyze the obtained DSC curves using the DSC-3 differential scanning calorimeter's accompanying software to obtain the enthalpy (ΔH) and peak temperature (Tp).

[0040] (4) FTIR measurement Dry starch samples were mixed with potassium bromide powder at a mass ratio of 1:100, and then thoroughly ground using an agate mortar. The uniformly ground starch sample was then pressed into a tablet and placed in the FTIR instrument slot for analysis. Specific parameter settings for the test were as follows: spectral resolution was set to 4 cm⁻¹. -1 The wavenumber range is set to 450cm. -1 -4000cm -1 Before testing, all starch samples were equilibrated in a 40℃ oven for 12 hours to maintain a uniform moisture content.

[0041] (5) Thermogravimetric analysis (TGA) was used to determine thermal stability. Accurately weigh 3 mg of starch sample and place it in a pre-weighed aluminum crucible, then transfer it to the thermogravimetric analyzer test chamber. Detection is performed under continuous high-purity nitrogen gas (flow rate 20 mL / min). The experiment uses a linear heating mode, increasing the temperature from 30℃ to 600℃ at a rate of 10℃ / min, while simultaneously obtaining differential thermogravimetric (DTG) data through mathematical processing.

[0042] (6) X-ray diffraction analysis The sample to be tested was evenly spread in the sample cell, and the crystal structure of the sample was determined using an X-ray diffraction (XRD) instrument. The test conditions were as follows: diffraction angle 2θ scanning range of 5°-60°, scanning speed of 7° / min, step size of 0.05°, accelerating voltage and current of 40kV and 40mA, respectively.

[0043] (7) X-ray photoelectron spectroscopy (XPS) The surface chemical composition of the samples was determined using a Thermo Scientific K-Alpha spectrometer. The excitation source was AlKα X-rays (hv = 1486.6 eV), the current was 16 mA, the voltage was 12 kV, the vacuum level in the analysis chamber was better than 5.0E-7 mBar, the full-spectrum scan energy was 100 eV, the narrow-spectrum scan energy was 20 eV, and the step size was 0.05 eV. The electron binding energy was corrected for the carbon contamination C1 (1s electron shell of C atoms) peak (284.8 eV). Data were processed using Avantage software.

[0044] (8) In vitro digestive performance test Weigh 200 mg of sample and disperse it in 2 mL of deionized water. Mix thoroughly and gelatinize for 20 min. Then, incubate the sample in a 37°C water bath shaker for 10 min. Add 4 mL of pepsin / hydrochloric acid solution (5 mg / mL) and shake for 30 min. Then, add 6 glass beads (3-4 mm in diameter) and 2 mL of sodium acetate buffer solution (0.5 mol / L, pH 5.2), mix well, and maintain for 30 min. Next, add 2 mL of a mixed enzyme solution (pancreatin, amylase, and invertase) to simulate digestion. At 0 min, 20 min, and 120 min, respectively, take 0.05 mL of the reaction solution and add it to 0.95 mL of ethanol solution (75%, v / v). Centrifuge at 10000 r / min for 5 min. Determine the glucose content in the supernatant using a glucose oxidase-peroxidase kit. Measure the absorbance of the test solution at 520 nm using a microplate reader. Calculate the RDS, SDS, and RS contents in the sample using the following formula: RDS (%) ×0.9 / TS×100%; SDS (%) )×0.9 / TS×100%; RS(%) (TS) RDS SDS) × 100%; G 20 and G 120The glucose content after 20 and 120 minutes of digestion are respectively; TS is the sample mass.

[0045] Example 1 A starch-quercetin complex enzymatic hydrolysate is prepared by the following steps: S1. Mix 10g of natural starch (glutinous rice starch, GS, amylopectin content 98%) with 100mL of distilled water, stir evenly with a glass rod, and gelatinize by heating and stirring in a water bath at 90℃ for 15min to obtain starch paste. S2. Add 2.5% quercetin to the starch paste, with the amount added based on the starch in S1. Stir for 1.5 hours to obtain starch-quercetin composite gel. S3. The starch-quercetin composite gel was allowed to stand at 25°C for 1 hour. Prussian blue enzyme purchased from Maclean's was added and enzymatically hydrolyzed in a water bath at 50°C for 5 minutes. The precipitate was collected, washed with anhydrous ethanol, and centrifuged 3 times to terminate the enzymatic hydrolysis reaction. The product was then freeze-dried to obtain the starch-quercetin composite enzymatic hydrolysate, denoted as GRS-Q5.

[0046] Example 2 The difference between Example 2 and Example 1 is that: The enzymatic hydrolysis time in step S3 is 15 min; The obtained starch-quercetin complex enzymatic hydrolysate was designated GRS-Q15.

[0047] Example 3 The difference between Example 3 and Example 1 is that: The enzymatic hydrolysis time in step S3 is 30 min; The obtained starch-quercetin complex enzymatic hydrolysate was designated GRS-Q30.

[0048] Example 4 The difference between Example 4 and Example 1 is that: The enzymatic hydrolysis time in step S3 is 60 min; The obtained starch-quercetin complex enzymatic hydrolysate was designated GRS-Q60.

[0049] Example 5 The difference between Example 5 and Example 1 is as follows: The enzymatic hydrolysis time in step S3 is 90 min; The obtained starch-quercetin complex enzymatic hydrolysate was designated GRS-Q90.

[0050] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that: Step S2 is skipped; In step S3, the object of enzymatic hydrolysis is starch paste, which is left to stand for 1 hour and the enzymatic hydrolysis time is 5 minutes; the remaining steps are the same as in Example 1. The resulting amylase hydrolysate was designated DBS-5.

[0051] Comparative Example 2 The difference between Comparative Example 2 and Comparative Example 1 is that: The enzymatic hydrolysis time in step S3 is 15 min; The resulting amylase hydrolysate was designated DBS-15.

[0052] Comparative Example 3 The difference between Comparative Example 3 and Comparative Example 1 is that: The enzymatic hydrolysis time in step S3 is 30 min; The obtained starch hydrolysate was designated DBS-30.

[0053] Comparative Example 4 The difference between Comparative Example 4 and Comparative Example 1 is that: The enzymatic hydrolysis time in step S3 is 60 min; The obtained starch hydrolysate was designated DBS-60.

[0054] Comparative Example 5 The difference between Comparative Example 5 and Comparative Example 1 is as follows: The enzymatic hydrolysis time in step S3 is 90 min; The obtained starch hydrolysate was designated DBS-90.

[0055] Comparative Example 6 The difference between Comparative Example 6 and Example 1 is as follows: Instead of proceeding to step S3, the starch-quercetin complex gel is obtained, washed and centrifuged with anhydrous ethanol, and then freeze-dried to obtain the starch-quercetin complex, which is GRS-Q.

[0056] Comparative Example 7 The difference between Comparative Example 7 and Comparative Example 6 is as follows: In step S2, the amount of quercetin added is 0.5%.

[0057] Comparative Example 8 The difference between Comparative Example 8 and Comparative Example 6 is as follows: In step S2, the amount of quercetin added is 1%.

[0058] Comparative Example 9 The difference between Comparative Example 9 and Comparative Example 6 is as follows: In step S2, the amount of quercetin added is 1.5%.

[0059] Comparative Example 10 The difference between Comparative Example 10 and Comparative Example 6 is as follows: In step S2, the amount of quercetin added is 2%.

[0060] Comparative Example 11 The difference between Comparative Example 11 and Comparative Example 6 is as follows: In step S2, the amount of quercetin added is 4%.

[0061] Performance testing 1. The branch chain length distribution of gelatinized starch, starch-quercetin complex (Comparative Example 6, GRS-Q), starch-quercetin complex enzymatic hydrolysates prepared in Examples 1-4, and starch hydrolysates prepared in Comparative Examples 1-4 was determined. The test results are as follows: Figure 1 As shown in Table 1. In this application, the gelatinized starch is obtained by directly freeze-drying the starch paste prepared in step S1 of Example 1, and is denoted as GRS.

[0062] Table 1 Results of branch chain length measurement

[0063] Chain length distribution refers to the statistical distribution of the degree of polymerization (DP) of side chains in amylopectin, i.e., the number of glucose units contained in the chain. Simply put, it's the proportion of chains of different lengths. In Table 1, DP6-12 refers to the percentage of side chains with a degree of polymerization of 6-12; DP13-24 refers to the percentage of side chains with a degree of polymerization of 13-24; DP25-36 refers to the percentage of side chains with a degree of polymerization of 25-36; DP37-76 refers to the percentage of side chains with a degree of polymerization of 37-76; and the average degree of polymerization refers to the average number of glucose units contained in each glucose chain constituting the product.

[0064] Depend on Figure 1As shown in Table 1, with the increase of debranching time (i.e., enzymatic hydrolysis time), the debranching time has no significant effect on the branch chain length distribution of starch hydrolysates, but it does affect the branch chain length distribution of starch-quercetin complex hydrolysates. At 30 min of enzymatic hydrolysis, the proportion of medium and long chains (i.e., DP25-36 and DP37-76) in GRS-Q30 is the highest, at 16.46% and 20.51%, respectively, which is higher than that of starch hydrolysate DBS-30 with the same debranching time. This indicates that the debranching treatment after combining natural starch with quercetin can increase the proportion of medium and long chains in starch branches, which is consistent with the results of the subsequent digestion experiments. Among them, there is a positive correlation between the change in branch chain length distribution and starch digestibility, including the decrease in long chain content corresponding to the decrease in resistant starch (RS) content, and the increase in short chain content and the increase in medium chain content corresponding to the increase in fast digestible starch (RDS) and slow digestible starch (SDS) content.

[0065] 2. The molecular weight of starch in gelatinized starch, starch-quercetin complex (Comparative Example 6, GRS-Q), starch-quercetin complex enzymatic hydrolysates prepared in Examples 1-5, and starch enzymatic hydrolysates prepared in Comparative Examples 1-5 was determined, and the results are shown in Table 2.

[0066] Table 2 Results of starch molecular weight determination

[0067] Debranching refers to the process of selectively hydrolyzing α-1,6-glycosidic bonds (i.e., branch points) in starch molecules using the specific enzyme pullulanase, while retaining α-1,4-glycosidic bonds (straight-chain linkages).

[0068] Table 2 shows that, compared with GRS and starch hydrolysates (natural starch gelatinized and hydrolyzed for different times), the number-average molecular weight of the starch hydrolysates is lower than that of natural starch, and the number-average molecular weight of the starch hydrolysates continues to decrease with increasing hydrolysis time. The branched structure of amylopectin is completely destroyed and "cut" into a bunch of linear chains of different lengths (i.e., debranching). Similarly, after the starch-quercetin complex GRS-Q was hydrolyzed, the number-average molecular weight of the starch in the resulting starch-quercetin complex hydrolysate gradually decreased with increasing hydrolysis time, but the number-average molecular weight of the starch increased significantly again at 90 min of hydrolysis, possibly because the starch "agglomerated".

[0069] 3. Fourier transform infrared spectroscopy (FTIR) was performed on gelatinized starch, quercetin, starch-quercetin complex (Comparative Example 6, GRS-Q), starch-quercetin complex enzymatic hydrolysates prepared in Examples 1-5, and starch enzymatic hydrolysates prepared in Comparative Examples 1-5. The FTIR spectra of starch-quercetin complex enzymatic hydrolysates (GRS-Q5, GRS-Q15, GRS-Q30, GRS-Q60, GRS-Q90), quercetin (Q), and starch-quercetin complex (GRS-Q) prepared in Examples 1-5 are shown in Figure 2(A). The FTIR spectra of starch enzymatic hydrolysates (DBS-5, DBS-15, DBS-30, DBS-60, DBS-90) and gelatinized starch (GRS) prepared in Comparative Examples 1-5 are shown in Figure 2(B). Further FTIR spectra of the above samples at 1047 cm⁻¹ were obtained. -1 Infrared intensity at 1022 cm -1 The infrared intensity at 1047 cm⁻¹ was compared, and the infrared ratio results are shown in Figure 2(C). The infrared ratio refers to the ratio of the infrared intensity at 1047 cm⁻¹ in the Fourier transform infrared spectrum of starch. -1 and 1022 cm -1 The ratio of the absorption peak intensities at two specific wavenumbers.

[0070] The helical structure, molecular chain conformation, and crystallinity of starch can be characterized by Fourier transform infrared spectroscopy (FTIR). Starch crystallinity is highest in the 3000-3600 cm⁻¹ range. -1 There is a broad and strong absorption peak, mainly due to the stretching vibrations of numerous hydroxyl groups on the starch molecular chain. As shown in Figure 2(B), compared to GRS, the starch complexed with quercetin exhibits a higher absorption peak in the 3000-3600 cm⁻¹ range. -1 The broadening of the absorption peak is due to the presence of multiple phenolic hydroxyl groups on the quercetin molecule. These phenolic hydroxyl groups form a new and stronger hydrogen bond network with the hydroxyl groups on the starch chain, the hydroxyl groups of quercetin itself, and water molecules in the system. As shown in Figure 2(A), compared with the starch-quercetin complex, the structure of the starch-quercetin complex hydrolysate is disrupted, and some characteristic peaks of quercetin gradually emerge. In the legend of Figure 2(C): 1047 cm⁻¹ -1 This is usually associated with highly ordered crystallization in starch, reflecting the tight arrangement of molecular chains; 1022 cm -1 Corresponding to the amorphous region of starch, the infrared ratio characterizes the loose or disordered structure of the molecular chain. The infrared ratio reflects the ratio of the two. The results showed that the branched structure of amylopectin originally formed ordered regions through close packing. After debranching, the linear segments rearranged due to thermodynamic instability, and some crystalline regions dissociated into an amorphous state. Compared with direct enzymatic hydrolysis, the infrared ratio of glutinous rice starch combined with quercetin and then enzymatically hydrolyzed decreased, and the disorder of starch increased. The infrared ratio also gradually decreased with the enzymatic hydrolysis time.

[0071] 4. Thermogravimetric analysis (TGA) was performed on gelatinized starch, starch-quercetin complex (GRS-Q), starch-quercetin complex enzymatic hydrolysates prepared in Examples 1-5, and starch enzymatic hydrolysates prepared in Comparative Examples 1-5. The TGA test results are shown in Figure 3(A). The differential thermogravimetric analysis (DTG) curve was obtained by first derivative of the TGA curve of starch. The DTG results are shown in Figure 3(B).

[0072] Thermal stability analysis (TGA) is an analytical method that measures the change in sample mass over temperature or time to represent the thermal stability of a sample. As shown in Figure 3(A), the sample weight loss occurs in two stages. The first small-scale mass loss step appears around 30-100℃, which is usually caused by the evaporation of moisture in the sample. The second stage of mass loss corresponds to the decomposition of starch, the main decomposition mechanism of which is the dehydration reaction between hydroxyl groups.

[0073] As shown in Figure 3(B), compared with gelatinized starch, the initial decomposition point of debranched starch (i.e., debranched natural starch or debranched starch) shifts to the left. Furthermore, the difference in the initial decomposition point between debranched starches from different debranching times is not significant. The debranching process thoroughly destroys the natural stable crystalline structure of starch granules, resulting in recrystallized regions with low integrity and numerous defects, making them prone to thermal decomposition. This result is corroborated by the changes in crystallinity observed in subsequent XRD and the porous morphology observed in subsequent SEM, jointly demonstrating that debranching treatment weakens the structural integrity of starch at multiple scales, from molecular to macroscopic, thereby leading to a decrease in its thermal stability. Compared to debranched starch-quercetin complex (i.e., starch-quercetin complex enzymatic hydrolysate), the initial decomposition point of the debranched starch-quercetin complex shifts to the left. However, the difference in the initial decomposition point between starch-quercetin complex enzymatic hydrolysates with different debranching times is not significant. This indicates that debranching treatment lowers the initial decomposition temperature (thermal stability) of the starch-quercetin complex. The degree of debranching has little effect on the initial decomposition temperature of the starch-quercetin complex, possibly because debranching treatment generates more easily decomposed linear starch. Compared to debranched starch without quercetin, the enzymatic hydrolysate obtained by debranching natural starch after quercetin conjugation exhibits higher thermal stability.

[0074] 5. The starch hydrolysate, gelatinized starch (GRS), and natural starch (GS) prepared in Comparative Examples 1-5 were subjected to DSC curve testing using a differential scanning calorimeter. The results are shown in Figure 4(A). The starch-quercetin complex and the starch-quercetin complex hydrolysate prepared in Examples 1-5 were subjected to DSC curve testing. The results are shown in Figure 4(B).

[0075] Differential scanning calorimetry (DSC) was used to study the thermodynamic behavior of starch, specifically the changes in its thermal properties during heating. These thermal property changes directly reflect the alterations in the starch double helix and crystalline structure. Factors influencing the thermodynamic properties of starch include starch molecular chain retrogradation, molar mass distribution, amylose content, degree of polymerization, and particle size.

[0076] As shown in Figure 4(A), the peak temperature of the GRS curve is 70.38℃, and the gelatinization enthalpy (ΔH) is 21.37 J / g. Figure 4(B) shows that after natural starch is combined with quercetin, the peak temperature is 121.02℃, and the ΔH is 34.68 J / g. This indicates that after natural starch is combined with quercetin, the initial temperature, peak temperature, and final temperature of the DSC curve generally shift towards higher temperatures. Compared to gelatinized starch, the gelatinization enthalpy of the starch-quercetin complex is significantly increased. This is mainly attributed to the formation of a more stable and complete complex between quercetin and starch. This complex differs from gelatinized starch, exhibiting a higher gelatinization enthalpy and peak temperature, making it difficult to gelatinize. Destroying this newly formed, highly ordered complex crystal structure requires additional energy, thus increasing the total area of ​​the DSC endothermic peak. After debranching with pullulanase, the gelatinization enthalpy and gelatinization temperature of the debranched starch-quercetin complex (i.e., starch-quercetin complex hydrolysate) increased compared to the unbranched form, indicating better stability of the complex structure. In contrast, the DSC curve of debranched natural starch did not show a melting peak, indicating that the debranching process led to the disordering of the natural starch molecular chains, forming a completely disordered, non-crystalline, fusible system.

[0077] 6. The multi-scale structure of natural starch (GS) and quercetin (Q) was observed by X-ray diffraction, and the results are shown in Figure 5(A); the multi-scale structure of starch-quercetin complex and starch-quercetin complex enzymatic hydrolysates prepared in Examples 1-5 was observed by X-ray diffraction, and the results are shown in Figure 5(B); the multi-scale structure of gelatinized starch and starch enzymatic hydrolysates prepared in Comparative Examples 1-5 was observed by X-ray diffraction, and the results are shown in Figure 5(C).

[0078] To investigate the effect of debranching on the multi-scale structure of starch, its long-range order was studied using X-ray diffraction. In the diffraction patterns, crystalline regions typically exhibit sharp diffraction peaks, reflecting the high order of molecular chain arrangement; while amorphous regions contribute diffuse broad peaks, reflecting the disordered state of molecular chains in this region. Figure 5(A) shows the XRD patterns of natural starch and quercetin. As can be seen from Figure 5(A), the XRD pattern represented by the black lines has peaks at 15.1°, 17°, 18°, and 23°, which is consistent with the type A crystal structure of natural starch. As shown in Figure 5(B), the starch-quercetin complex has peaks at 10.7°, 12.4°, and 27.5°, while quercetin has peaks at 9.4°, 13.1°, 14.1°, 16.9°, 22.0°, and 26.5°. After debranching with pullulanase, the starch-quercetin complex showed multiple diffraction peaks. Compared with the undebranched starch-quercetin complex, the crystallinity of the debranched starch-quercetin complex increased significantly to 15.30%. This may be because quercetin occupies the steric hindrance of the amorphous region of starch, allowing more segments to participate in the formation of the crystalline region. Figure 5(C) shows the XRD pattern of the starch hydrolysate. After the natural starch was gelatinized, the characteristic peaks disappeared, and the pattern showed a "bread peak". Moreover, after the natural starch was debranched with pullulanase for different times, no diffraction peaks appeared in the samples. The reason may be that the starch molecular chains did not have time to rearrange after debranching and no new crystallization region was formed. This result is consistent with the DSC results.

[0079] 7. Based on the distribution of branch chain lengths, GRS-Q30 and GRS-Q60 were selected for X-ray photoelectron spectroscopy analysis.

[0080] Specifically, X-ray photoelectron spectroscopy analysis was performed on gelatinized starch, starch-quercetin complex hydrolysate prepared in Examples 3-4, starch-quercetin complex, and starch hydrolysate prepared in Comparative Examples 3-4. The XPS spectra are shown in Figures 6(A), 6(B), 6(C), and 6(D).

[0081] X-ray photoelectron spectroscopy (XPS) analysis primarily analyzes the starch structure by measuring the chemical states of carbon (C) and oxygen (O) atoms. In Figure 6(A), the peaks at 531 eV and 285 eV are caused by C and O elements, respectively. Compared with gelatinized starch, the starch-quercetin complex, starch hydrolysate, and starch-quercetin complex hydrolysate did not introduce new peaks. As shown in Figure 6(B), compared with GRS, DBS-30, and DSB-60, gelatinization + enzymatic hydrolysis increased the O content of natural starch. This is mainly because the debranching process led to a drastic reorganization of the starch molecule surface structure and properties, resulting in the enrichment of hydrophilic oxygen atoms on the sample surface. Compared with gelatinized starch, the main reason for the increased O content in the starch-quercetin complex is that the introduction of quercetin significantly altered the molecular arrangement and interactions on the starch surface, leading to the enrichment of oxygen-rich polar groups (such as hydroxyl and carbonyl groups) on the outermost layer of the sample; at the same time, quercetin itself also contributed additional oxygen atoms. After debranching, the content of O decreased in the starch-quercetin complex, indicating that pullulanase debranching triggered a surface recombination effect in the starch-quercetin complex system.

[0082] As shown in Figure 6(C), the high-resolution C1s spectrum, and Figure 6(D), the high-resolution O1s spectrum, show three characteristic peaks in the C1s spectrum and one characteristic peak in the O1s spectrum. These peaks, from right to left, correspond to the following chemical groups: CC / CH (C1), CO (C2), COC (C3), and O2 (C=O). After starch is combined with quercetin, the CC / CH peak is significantly enhanced. This is because a hydrophobic aromatic ring and a polar carbonyl group from quercetin are introduced into the starch surface or molecular chain network, and may "shield" some of the hydrophilic groups of starch through interactions such as hydrogen bonds. After debranching the starch-quercetin complex, the CC / CH peak is enhanced, while the CO peak is weakened. This is because the mobility of the short chains increases after debranching, making it easier for the hydrophobic portion to accumulate on the surface during drying and film formation. By severing the branch points and releasing the short chains, the flexibility and rearrangement ability of the molecular chains are increased, thereby intensifying surface hydrophobicity and improving digestibility.

[0083] 8. Scanning electron microscopy was performed on quercetin, and the results are as follows: Figure 7 As shown, the left image is magnified to 1000x and the right image to 2000x. Scanning electron microscopy was used to observe gelatinized starch, starch-quercetin complex, starch hydrolysates prepared in Comparative Examples 1-5, and starch-quercetin complex hydrolysates prepared in Examples 1-5. Figure 8 and Figure 9 As shown, Figure 8 The magnification is 1000. Figure 9 The magnification is 2000.

[0084] like Figure 7 As shown, quercetin is clearly observed to mainly exist as needle-like crystals; from Figure 8 and Figure 9 It can be seen that GRS particles exhibit an irregular polygonal structure with clearly defined edges and corners, a smooth surface without obvious cracks, and a particle size distribution range of approximately 3-10 μm. After pullulanase debranching treatment, the diameter and surface morphology of natural starch granules undergo significant changes, from... Figure 8 It can be seen that the debranched starch exhibits a microscopic morphology highly similar to that of a honeycomb briquette, with its surface covered with dense honeycomb-like pores, forming an interconnected three-dimensional porous structure. This change is likely due to pullulanase specifically cleaving α-1,6 glycosidic bonds during the debranching process, disrupting the molecular skeleton and crystalline regions of starch. This causes its dense granular structure to collapse and recombine into a loose, porous morphology. Figure 9 It is known that the starch-quercetin complex undergoes similar structural changes before and after debranching, providing a possibility for regulating digestibility. With this structural change, quercetin molecules, originally embedded in the starch helical cavity and amorphous region, are gradually released due to the loss of support and shielding, migrating to the surface of the newly formed giant pores, thus achieving physical "exposure." This exposure process directly enhances the bioaccessibility of quercetin and its interaction with the external environment. Before debranching, most of the quercetin in the starch-quercetin complex is tightly embedded by starch molecules, making it difficult for its biological activity or reaction sites to come into contact with the external environment. As enzymatic hydrolysis time increases, the complex structure of the starch-quercetin complex is gradually destroyed, especially at 60 min and 90 min, where the quercetin particles show significant disruption.

[0085] 9. The in vitro digestion characteristics of gelatinized starch, starch-quercetin complex, starch-quercetin complex enzymatic hydrolysates prepared in Examples 1-3, and starch enzymatic hydrolysates prepared in Comparative Examples 1-3 were tested, and the results are shown in Table 3.

[0086] Table 3 Results of in vitro digestion tests

[0087] In Table 3, the differences between data with different superscript letters in the same column (data in Mean±SD form) were statistically significant (P<0.05). Here, RDS refers to rapidly digestible starch; SDS refers to slowly digestible starch; and RS refers to resistant starch.

[0088] To investigate the effect of debranching on the in vitro digestibility of starch, this application determined the contents of rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) in starch and its debranching products. The changes in the contents of these components were compared and analyzed to reveal the effect of debranching. Table 3 shows that, for gelatinized starch, the debranched natural starch (i.e., starch hydrolysate) showed a decrease in RDS content and an increase in SDS and RS content compared to gelatinized starch. This may be because debranching, as an auxiliary structural modification, can generate more amylose single-helix chains, inducing the formation of ordered V-shaped crystal nuclei and reducing starch digestibility. Based on the analysis in Table 3, for starch-quercetin complexes, the debranched hydrolysate showed a decrease in RDS content and an increase in SDS and RS content compared to the undebranched starch-quercetin complex. This may be because quercetin inhibits α-amylase, resulting in a higher proportion of medium and long chains at a hydrolysis time of 30 min, leading to a decrease in RDS content and an increase in SDS and RS content.

[0089] 10. The complexation degree of starch-quercetin complexes with different quercetin contents was detected. The test method was as follows: 50 mg of starch-quercetin complex was dispersed in 5 mL of deionized water and centrifuged at 4000 r / min for 15 min. 0.1 mL of the supernatant was mixed with 0.2 mL of 6% (w / v) sodium carbonate solution and reacted under light-protected conditions for 20 min. The absorbance was measured at 725 nm to obtain the quercetin content in the supernatant, denoted as M1. The amount of quercetin added to the 50 mg starch-quercetin complex was denoted as M0, and the complexation rate of quercetin was calculated as follows: Complexation rate (%) = (1-M1 / M0)×100; the test results are as follows. Figure 10 As shown.

[0090] Depend on Figure 10 It can be seen that the complexation degree of the starch-quercetin complex exhibits a trend of first increasing and then decreasing. The complexation degree is highest at 1.5% quercetin (Comparative Example 9), reaching 21%. In the lower stages of quercetin addition, starch provides a large number of vacant hydrophobic cavities and binding sites, allowing each quercetin molecule to find a suitable site to bind. Therefore, as the amount of quercetin increases, the complexation degree increases linearly until the effective binding sites of starch molecules are almost saturated. At this point, further increases in quercetin lead to very slow or even cessation of the increase in complexation degree. This point of cessation can be considered the "equilibrium point" or "saturation point" of the complexation reaction. When the amount of quercetin exceeds the maximum inclusion capacity of starch, the complexation degree begins to decrease.

[0091] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. A method for preparing a starch-quercetin complex enzymatic hydrolysate, characterized in that, The preparation method of the starch-quercetin complex enzymatic hydrolysate includes the following steps: S1. Mix starch and water, heat to gelatinize, and obtain starch paste; S2. Mix the starch paste and quercetin, stir, and obtain a starch-quercetin composite gel; S3. Under the enzymatic hydrolysis temperature and pH conditions of pullulanase, the starch-quercetin composite gel and pullulanase are mixed and subjected to enzymatic debranching reaction to obtain starch-quercetin composite hydrolysate.

2. The method for preparing the starch-quercetin complex enzymatic hydrolysate as described in claim 1, characterized in that, In step S1: The temperature for heating and gelatinizing is 85-95℃; and / or, The heating and gelatinization time is 10-20 minutes.

3. The method for preparing the starch-quercetin complex enzymatic hydrolysate as described in claim 1, characterized in that, In step S2: The mass ratio of quercetin to starch is 1-5:100; and / or, The stirring time is 1-2 hours.

4. The method for preparing the starch-quercetin complex enzymatic hydrolysate as described in claim 1, characterized in that, In step S3: The enzymatic hydrolysis temperature is 45-55℃; and / or, The enzymatic hydrolysis pH is 4.5-5.5; and / or, Add 10-30 U pullulanase per 1g of starch; and / or, The enzymatic debranching reaction takes 5-90 minutes.

5. The method for preparing the starch-quercetin complex enzymatic hydrolysate as described in claim 1, characterized in that, Step S3 includes: Under the enzymatic hydrolysis temperature and pH conditions of pullulanase, starch-quercetin composite gel and pullulanase were mixed and subjected to enzymatic debranching reaction. The precipitate was collected, washed with anhydrous ethanol, centrifuged, and freeze-dried to obtain starch-quercetin composite hydrolysate.

6. A starch-quercetin complex enzymatic hydrolysate, characterized in that, The starch-quercetin complex enzymatic hydrolysate is prepared by the method for preparing starch-quercetin complex enzymatic hydrolysate according to any one of claims 1 to 5; the starch-quercetin complex enzymatic hydrolysate comprises a starch derivative and quercetin bound to the starch derivative through a V-shaped single-helix inclusion structure: The branched chain length distribution of the starch derivative is as follows: the proportion of branched segments with a degree of polymerization of DP25-36 is ≥16%, and the proportion of branched segments with a degree of polymerization of DP37-76 is ≥20%. In the starch-quercetin complex enzymatic hydrolysate: the mass percentage of rapidly digestible starch is less than 55%, and the mass percentage of resistant starch is greater than 30%.

7. The application of a starch-quercetin complex enzymatic hydrolysate in low glycemic index foods, slow-release energy foods, or functional health foods, characterized in that, The starch-quercetin complex enzymatic hydrolysate includes the starch-quercetin complex enzymatic hydrolysate prepared by the method described in any one of claims 1 to 5, or the starch-quercetin complex enzymatic hydrolysate as described in claim 6.

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