A method for increasing the digestion resistance of debranched corn starch

Abstract

The application discloses a method for improving the anti-digestibility of debranched corn starch, and relates to the technical field of starch processing. The method comprises the following steps: mixing a debranched starch solution, a chitosan solution and a tannic acid solution to carry out a mixing reaction, and centrifuging to obtain a precipitate; and drying and grinding the precipitate to obtain a debranched starch-chitosan-tannic acid compound. The method provided by the application shows the potential of effectively improving the anti-digestibility of starch through the synergistic effect of multiple components by constructing a ternary composite system, provides a new feasible path for developing low-GI staple food, sugar-controlled food and functional materials for stably delivering polyphenols, and provides an important enlightenment for constructing a starch-based composite system with a specific structure-digestion behavior.

Description

Technical Field

[0001] This invention relates to the field of starch processing technology, and in particular to a method for improving the digestibility of debranched corn starch. Background Technology

[0002] Starch, as the primary source of energy in the human diet, plays an indispensable role in maintaining human life and in food production. However, natural starch is readily recognized and hydrolyzed by α-amylase and glucosylamylase in the upper digestive tract, leading to a rapid spike in postprandial blood glucose levels. Epidemiological studies have shown a significant positive correlation between long-term consumption of such high glycemic index (GI) carbohydrate diets and the development of chronic metabolic syndromes such as obesity, type II diabetes, and cardiovascular disease. Therefore, modifying the fine structure and digestive kinetics of starch to create resistant starch (RS) with anti-digestive properties is crucial for improving dietary health.

[0003] Among numerous modification strategies, enzymatic debranching-recrystallization technology stands out due to its green and controllable nature. Pullulanase can specifically cleave the α-1,6-glycosidic bonds in amylopectin molecules, releasing a large amount of short-chain glucans. Under low-temperature induction, these short-chain molecules can overcome steric hindrance and undergo self-assembly and orderly arrangement driven by intermolecular hydrogen bonds, forming a dense double-helix crystalline structure, namely resistant starch type III (RS3). However, the surface of single debranched starch (DBS) microspheres still lacks sufficient steric hindrance to completely block the adsorption and penetration of enzyme proteins, limiting its application potential as a long-acting sugar-controlling ingredient in complex food systems. Summary of the Invention

[0004] The purpose of this invention is to provide a method for improving the digestibility of debranched corn starch, thereby addressing the problems existing in the prior art. This method can effectively improve the digestibility of debranched corn starch, providing technical support for the development of low-GI starch-based functional foods.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for improving the digestibility of debranched corn starch chains, comprising the following steps: The debranched starch solution, chitosan solution, and tannic acid solution were mixed and reacted, and then centrifuged to obtain the precipitate. The precipitate was dried and ground to obtain a debranched starch-chitosan-tannic acid complex.

[0006] Furthermore, the temperature of the mixing reaction is 4°C.

[0007] Furthermore, the mixing reaction time is 24 hours.

[0008] Furthermore, the pH value of the mixed reaction is 4.8~5.0.

[0009] Furthermore, the concentration of the chitosan solution is 1-2 wt%.

[0010] Preferably, the concentration of the chitosan solution is 1 wt%.

[0011] Furthermore, the debranched starch is obtained by enzymatic hydrolysis of gelatinized corn starch using pullulanase.

[0012] The present invention also provides a debranched starch-chitosan-tannic acid complex prepared according to the above method.

[0013] The present invention also provides the application of the above-mentioned debranched starch-chitosan-tannic acid complex in the preparation of low-GI starch-based functional foods.

[0014] The present invention also provides a low-GI starch-based functional food, comprising the above-mentioned debranched starch-chitosan-tannic acid complex.

[0015] The present invention discloses the following technical effects: This invention utilizes self-assembly technology to construct debranched starch-chitosan-tannic acid (DBS-CS-TA) ternary composite microspheres, aiming to enhance the digestibility of starch. Multiscale structural characterization reveals that the introduction of tannic acid (TA) significantly alters the assembly mode of the DBS-CS binary system, acting as a "molecular bridge" to construct a broad hydrogen bond network between the DBS and CS interfaces. Meanwhile, the addition of chitosan (CS) significantly modulates the microstructure of the DBS-TA system; with increasing CS concentration, the resulting steric hindrance and competitive hydrogen bonding intensify, leading to a decrease in the system's relative crystallinity and short-range order (I0.05). 1047 / 1022 The enthalpy of gelatinization (ΔH) gradually decreased. Although the impaired integrity of the crystalline regions led to a reduction in the gelatinization enthalpy (ΔH), the dense cross-linked network among the three components effectively constrained the thermal motion of the complex skeleton, resulting in an overall increase in the thermal phase transition temperature. This confirms that the complex sacrifices local orderliness for enhanced overall structural rigidity. Molecular dynamics simulations further corroborate this, showing that the anchoring effect of TA significantly suppressed dynamic fluctuations in the skeleton and maintained the spatial compactness of the conformation. In vitro digestion experiments showed that the DBS-CS-TA system had a high resistant starch content, demonstrating that moderate multi-component cross-linking can effectively achieve precise intervention in starch digestion rate. This invention reveals the regulatory mechanism of multi-component interactions on starch structure and digestive behavior, providing technical support for the development of low-GI starch-based functional foods. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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 these drawings without creative effort.

[0017] Figure 1 SEM images of different samples; where A: WMS; B: DBS; C: DBS-0CS-TA; D: DBS-1CS-0TA; E: DBS-1CS-TA; F: DBS-1.5CS-TA; G: DBS-2CS-TA; scale bar is 10 μm. Figure 2 CLSM plots for different samples; where A: WMS; B: DBS; C: DBS-0CS-TA; D: DBS-1CS-0TA; E: DBS-1CS-TA; F: DBS-1.5CS-TA; G: DBS-2CS-TA; scale bar is 20 μm. Figure 3 The ζ-potential (A) and particle size distribution (B) of different samples are shown. Figure 4 XRD patterns (A) and FTIR patterns (B) of different samples; Figure 5 Thermogravimetric analysis (TGA) plots for different samples are shown below; where A: WMS; B: DBS; C: DBS-0CS-TA; D: DBS-1CS-0TA; E: DBS-1CS-TA; F: DBS-1.5CS-TA; G: DBS-2CS-TA. Figure 6 DSC analysis chromatograms for different samples; Figure 7 Figures showing the in vitro digestion characteristics (A) and digestion kinetics (B) of different samples; Figure 8 The diagram shows the molecular docking of debranched corn starch / chitosan / tannic acid complexes. A: Surface binding sites of the DBS-CS complex; B: 3D binding conformation and hydrogen bonding of the DBS-CS complex; C: 2D interaction diagram of the DBS-CS complex; D: Surface binding sites of TA in the DBS-CS system; E: 3D binding conformation and hydrogen bonding of the DBS-CS-TA complex; F: 2D interaction diagram of the DBS-CS-TA complex. Figure 9The graphs show the molecular dynamics trajectories of different samples; where A represents the RMSD curves of DBS, CS, and DBS-CS; B represents the RMSD curves of TA, DBS-CS, and DBS-CS-TA; C represents the Rg curves of DBS, CS, and DBS-CS; and D represents the Rg curves of TA, DBS-CS, and DBS-CS-TA. Figure 10 This is a schematic diagram of the structural model and anti-digestion mechanism of the debranched corn starch / chitosan / tannic acid complex. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] The experimental materials involved in the following examples are as follows: Waxy corn starch (WMS) (amylose content 2.2%) was purchased from Shandong Huanong Special Corn Development Co., Ltd.; pullulanase (2250 ASPU / mL) was purchased from Danisco (Denmark); chitosan (CAS: 9012-76-4, degree of deacetylation >90%) was purchased from Shandong Aokang Biotechnology Co., Ltd.; tannic acid (purity >95%) was purchased from Sigma-Aldrich; all other reagents were of analytical grade.

[0024] Example 1 1. Preparation of debranched starch-chitosan-tannic acid ternary complex A 10% (w / v) waxy corn starch slurry was prepared, and the pH was adjusted to 5.0 using 0.01 mol / L acetate buffer. The starch slurry was pregelatinized at 95 °C for 30 min, followed by high-temperature and high-pressure heat treatment at 121 °C for 20 min to further promote molecular chain extension and ensure complete gelatinization. After cooling to 58 °C, pullulanase was added and enzymatically hydrolyzed at an incubation temperature for 8 h. The reaction was then terminated by treatment at 121 °C for 20 min. After centrifugation at 4000 rpm for 10 min, the supernatant was collected and cooled to room temperature to obtain the debranched starch (DBS) solution.

[0025] Chitosan (CS) solutions with mass fractions of 0%, 1%, 1.5%, and 2% were prepared using 1% acetic acid. Equal volumes of DBS solutions were mixed with CS solutions of different concentrations, and tannic acid (TA) solution was added to make the final TA addition amount 4% of the dry weight of debranched starch. The pH of the mixture was adjusted to 4.8–5.0 with 0.5 mol / L NaOH, and the mixture was dynamically incubated at 4°C for 24 h. Utilizing the synergistic effect of low-temperature retrogradation of debranched starch and electrostatic-hydrogen bonding, multi-component ordered co-assembly was induced, and composite microspheres were constructed in situ. The reaction solution was centrifuged (4000 rpm, 10 min) to separate the precipitate. The precipitate was pre-frozen at -80℃, then freeze-dried under vacuum, ground, and passed through a 100-mesh sieve to obtain debranched starch-chitosan-tannic acid complexes with different ratios. The complexes were named DBS-0CS-TA, DBS-1CS-TA, DBS-1.5CS-TA, and DBS-2CS-TA according to the chitosan solution concentrations of 0%, 1%, 1.5%, and 2%, respectively.

[0026] TA solution was added to the DBS solution to make the final TA addition amount 4% of the starch mass. The pH of the mixture was adjusted to 4.8-5.0 with 0.5 mol / L NaOH, and the reaction was carried out at 4℃ for 24 h. The reaction solution was centrifuged (4000 rpm, 10 min) to separate the precipitate. The precipitate was freeze-dried, ground, and passed through a 100-mesh sieve to obtain the debranched starch-tannic acid complex, named DBS-0CS-TA.

[0027] A blank control was used for a sample that had only undergone debranching treatment without the addition of CS and TA.

[0028] 2. Characterization of the complex 2.1 Scanning electron microscopy (SEM) morphological analysis of the complex The morphology of the samples was observed using a scanning electron microscope (SU1510, Hitachi, Japan). The samples were laid on double-sided adhesive tape and sputtered with gold. The morphology of the samples was observed under an accelerating voltage of 5 kV, and their morphological features were recorded at magnifications of 500-5000x.

[0029] like Figure 1 As shown, WMS particles exhibit a polyhedral characteristic with sharp edges, no pores or cracks, and a smooth surface. Their particle size ranges from 10 to 20 μm. After enzymatic debranching, their surface structure undergoes significant changes. DBS samples mostly appear as regular, tiny spherical particles with nanometer-sized grains, and the overall structure of DBS is relatively compact. It is worth noting that the structural differences of the selected starch raw materials before debranching also affect the self-assembly behavior of the particles after debranching. Studies have shown that longer amylose chains in the raw materials may penetrate amorphous regions during debranching, disrupting the orderly stacking of short-chain dextran, thus leading to particle aggregation and the formation of large, irregular blocky structures. Compared to DBS-1CS-0TA, DBS-0CS-TA exhibits less aggregation and a more uniform particle size. For the ternary composite system, with the increase of chitosan content, the amount of chitosan adhering to the particle surface gradually increases, the bonding between particles becomes tighter, and the particle aggregation phenomenon becomes more pronounced.

[0030] 2.2 CLSM morphological analysis of the complex The morphology and structure of the complex were observed using a laser confocal microscope (LSM 980, Germany). The sample was dispersed in deionized water, mixed with Rhodamine B (20 μL, 2 mg / mL) in 5 mL for staining, and observed under a laser confocal scanning microscope. The excitation wavelength was 546 nm, and the emission wavelength was 568 nm.

[0031] like Figure 2 As shown, DBS volume is significantly smaller than WMS, but both have relatively uniform volume distribution. The addition of TA weakens the aggregation of the complex, while the addition of CS and TA makes the volume distribution of the complex more extensive. Moreover, the volume of the complex increases with the increase of CS, which is consistent with the results observed by SEM.

[0032] 2.3 Particle size and ζ-potential determination The particle size of the samples was determined using a laser particle size analyzer (Bettersize 2600, Dandong, China). The refractive indices of water and samples were set to 1.33 and 1.52, respectively. The zeta potential was determined using a potentiometer (BeNano 90 Zeta, China). All tests were performed at 20 °C.

[0033] like Figure 3 As shown in Figure A, untreated starch carries a negative charge, which decreases after debranching. The addition of TA increases the negative potential of the sample, indicating that TA recombines with DBS and adsorbs onto the DBS surface, significantly altering the surface electrical properties of the particles. The introduction of CS imparts a positive charge to the particle surface, originating from the protonated amine groups (-NH3) in the CS molecule. + This causes the zeta potential to change from negative to positive. When 1% CS was added and combined with TA (DBS-1CS-TA), the positive potential decreased slightly compared to the sample with CS alone (DBS-1CS-0TA), presumably because partial electrostatic association formed between TA and CS weakened its surface positive charge. With further increases in the amount of CS added, the surface positive potential of the composite particles increased significantly, showing an enhanced charge trend, reflecting an increase in the density of protonated amine groups in the system and an enhanced coverage of CS on the particle surface.

[0034] like Figure 3 As shown in Figure B, the particle size of DBS is significantly reduced after debranching, and its D... 50 The particle size decreased from 15.29 ± 0.23 μm in WMS to 11.74 ± 0.02 μm, indicating that the short-chain dextran formed after long-chain breakage can self-assemble into smaller and more compact microsphere structures. After the addition of TA, the particle size further decreased to 5.19 ± 0.14 μm. This is because TA increases the absolute value of the particle surface potential, enhancing electrostatic repulsion and thus reducing microsphere aggregation. For the ternary system, with the increase of CS addition, the particle size distribution of the sample gradually broadened, and D... 50 The volume also increases accordingly, possibly because as the amount of chitosan added increases, more and more chitosan adheres to the surface of the starch microspheres, and TA further cross-links with CS and DBS through electrostatic interactions and hydrogen bonds, leading to particle aggregation and increased volume. This indicates that the cross-linking effect among the three components in the system is greater than the electrostatic repulsion effect brought about by their composite composition.

[0035] 2.4 X-ray diffraction (XRD) crystallography of the complex X-ray diffraction patterns were measured using an X-ray diffractometer (SmartLab, Shimadzu, Japan) under conditions of 40 kV voltage and 40 mA current using a Cu-Ka radiation detector (λ = 0.154 nm). Samples were scanned in the (2θ) range of 4°–45° at a scan rate of 2° / min with a step size of 0.02°. Pattern processing was performed using MDI jade 6.0, and the relative crystallinity (%) was calculated using the following formula: ; Where: RC - relative crystallinity, %; Ac - area of ​​crystalline region on X-ray diffraction pattern; Aa - area of ​​amorphous region on X-ray diffraction pattern.

[0036] Figure 4 Table A and Table 1 show the X-ray diffraction patterns and relative crystallinity of different samples. WMS exhibits a typical type A crystal form with characteristic diffraction peaks at 2θ = 15.1°, 17.2°, 18.0°, and 23.0°. After debranching, the crystal form transforms into type B, with main peaks near 17.2°, 22.5°, and 24°, and the crystallinity decreases to 38.84 ± 0.57%. Adding only TA further reduces the relative crystallinity, possibly because the polyphenolic hydroxyl groups of TA compete with starch segments for hydrogen bond sites, disrupting the orderly stacking of the double helix and thus weakening the crystal structure. Adding CS alone significantly increases crystallinity. Studies have shown that CS can form multi-point hydrogen bonds with starch hydroxyl groups through its amino groups, promoting the orderly arrangement of short-chain starch and the formation of stable crystalline regions, while also enhancing the structural compactness and template-induced effect of the self-assembled system. However, when CS and TA are added simultaneously, the crystallinity decreases. This is likely due to the formation of numerous hydrogen bonds and electrostatic associations between the two, leading to excessive cross-linking and weakening the orderly rearrangement ability of the starch chains. As the amount of CS added continues to increase, the excessive cross-linking and steric hindrance further inhibit the orderly arrangement of the starch chains, resulting in a continuous decrease in crystallinity.

[0037] Table 1. Crystallization type, relative crystallinity, and short-range order of debranched corn starch / chitosan / tannic acid complexes

[0038] 2.5 Fourier Transform Infrared Spectroscopy (FT-IR) Analysis The sample was mixed with KBr (mass ratio 1:150), repeatedly ground using an agate mortar and pestle, and then compressed into tablets using a tablet press. FTIR spectra were collected (Magna-IR IS50, Bruker, Germany), with 32 scans at a resolution of 4 cm⁻¹. -1 The range is 400 to 4000 cm. -1 .

[0039] Infrared spectroscopy is an important tool for studying the structure and functional group changes of starch molecules, and can be used to analyze intermolecular interactions in complexes. For example... Figure 4 As shown in Figure B, all samples were at 3400 cm⁻¹ -1 The broad absorption band at 2930 cm⁻¹ -1 The absorption peaks at [values ​​missing] represent the stretching vibrations caused by -OH and -CH in the glucose unit, respectively. Compared with WMS, the hydroxyl absorption peak of the debranched sample changed from 3421 cm⁻¹ to [value missing]. -1 Redshifted to 3407 cm -1 Furthermore, the absorption intensity was significantly enhanced, indicating that the debranching treatment exposed more free hydroxyl groups. CS at 1591 cm⁻¹ -1 The sample exhibits an absorption peak for protonated amine groups, but this peak disappears in the DBS-1CS-0TA sample, indicating that the amine groups may bind to the hydroxyl groups on the starch molecule surface through electrostatic or hydrogen bonding, altering the vibrational environment of the amine groups. Furthermore, compared to DBS, DBS-1CS-0TA shows extended peaks for -OH and -NH (4000-3300 cm⁻¹). -1 The broadening of the peak indicates that the hydroxyl groups in debranched starch interact with the amino groups of chitosan through hydrogen bonds. Further addition of TA resulted in a continued broadening of the absorption band in this region, suggesting that the polyphenolic hydroxyl groups in TA also form a hydrogen bond network with chitosan and starch molecules, enhancing the intermolecular association strength of the system.

[0040] In the infrared spectrum, 1047 cm⁻¹ -1 Absorption peaks typically correspond to the short-range ordered structure of starch molecules, while 1022 cm⁻¹... -1 The absorption peaks primarily reflect the vibrational characteristics of the amorphous region; therefore, their ratio I 1047 / 1022 It is often used as an indicator to evaluate the short-range orderliness of starch; a higher value indicates a more regular arrangement of local segments. As shown in Table 1, debranching treatment has no significant effect on the short-range orderliness of starch; in contrast, whether only TA or only CS is added, I... 1047 / 1022 Both were significantly lower than DBS (P<0.05), indicating that both interfere with the local ordered stacking of short chains through multi-point hydrogen bonding or coating, making the short-range structure tend to be loose. In the ternary composite system, the crystallinity of DBS-1CS-TA was higher than that of DBS-0CS-TA, but I 1047 / 1022 The further decrease indicates that the introduction of CS (Chemical Solids) is beneficial to the growth of crystalline regions on a larger scale, and on a local scale, it participates in the inter-chain interactions of starch with TA (Ta), redistributing the hydrogen bonds between chain segments and making the short-range structure more loose. With increasing CS content, the interactions between DBS-CS-TA become more complex, and the local structure tends towards amorphous. At the same time, the steric hindrance caused by excessive CS around the chain segments also restricts the formation of short-range ordered structures, thus affecting I... 1047 / 1022 Further reduction.

[0041] 2.6 Thermogravimetric analysis (TGA) of the complex The thermal properties of the sample were analyzed using a thermogravimetric analyzer (Shimadzu TGA-50, Kyoto, Japan). Approximately 5.0 mg of sample was weighed and placed in an alumina crucible. The sample was heated from 30 °C to 800 °C at a heating rate of 10 °C / min under a nitrogen atmosphere, and the mass loss of the sample was recorded as a function of temperature.

[0042] Thermogravimetric analysis was used to analyze the thermal degradation of the samples, such as... Figure 5 All samples exhibited a typical two-stage weight loss characteristic during thermal degradation. The first stage (T < 125 °C) involved the release of moisture and volatile components, while the second stage (approximately 200-400 °C) involved main chain thermal decomposition. Specifically, WMS and DBS showed weight loss rates of 10.24% and 8.46%, respectively, in the first stage. With increasing CS content in the composite system, the weight loss rate in the first stage gradually decreased, which may indicate that the dense cross-linked network within the composite restricted the free migration of water molecules, improving the hydrothermal stability of the material. In the second stage, the main decomposition peak for all samples was located between approximately 341-345 °C. However, in the ternary system, with increasing CS content, T... max The decreasing trend is attributed to the decrease in crystallinity and the increase in the proportion of amorphous regions with lower thermal stability in the system, which offsets the thermal stabilization effect brought about by some crosslinking.

[0043] 2.7 DSC analysis of the complex Accurately weigh 4.0 mg of sample and place it in a DSC crucible. Add 10 μL of ultrapure water, stir and mix well, seal and store at room temperature for 6–8 h. The blank control is an empty crucible. Set the heating conditions to 25–120 °C at a heating rate of 10 °C / min. Analyze the obtained DSC curves using analytical software to obtain the enthalpy (ΔH), onset temperature (To), peak temperature (Tp), and termination temperature (Tc).

[0044] Starch gelatinization is a physical transformation process. During this process, the double helix structure of amylopectin dissociates, and its crystalline structure transforms into an amorphous structure. The energy required for the double helix structure to disintegrate is defined as enthalpy change (ΔH). Differential scanning calorimetry (DSC) can be used to characterize the structural stability and crystalline integrity of starch systems during heating. Figure 6As shown in Table 2, compared with WMS, DBS showed a significant increase in Tp and a marked increase in ΔH, indicating that the debranched short-chain starch can reassemble into a denser double helix structure, enhancing the integrity of the crystalline regions. When only TA was added, both To and Tp increased, but ΔH decreased significantly. This may be because the polyphenolic hydroxyl groups of TA bind to starch segments through multi-point hydrogen bonds, increasing the degree of binding between segments and making the structure more difficult to relax in the initial heating stage; on the other hand, it partially disrupts the original crystalline regions, causing the system to undergo an endothermic transformation from a regular crystal to a partially amorphous structure, reducing the energy required for crystalline region destruction. When only CS was added, ΔH also decreased, consistent with the weakening of short-range order. Because CS forms a new hydrogen bond network with the starch chains, it interferes with the regular stacking of short chains, making the crystalline regions tend to be looser. Even though the crystallinity is improved macroscopically, its internal structure is still not dense enough. In the ternary composite system, with increasing CS content, To, Tp, and Te all show an increasing trend, while ΔH continuously decreases, consistent with the decreasing trend of crystallinity and short-range order revealed by XRD and FTIR. This indicates that when CS and TA coexist, a more complex multi-point interaction network is formed within the system, enhancing the binding force between chain segments and increasing the onset temperature required for thermal transformation. However, the steric hindrance introduced by excessive CS and the hydrogen bonds formed competitively with TA and starch segments disrupt the orderly stacking within the crystalline regions, leading to a decrease in crystalline region quality and greater disorder, ultimately manifested as a gradual decrease in ΔH with increasing CS content.

[0045] Table 2. DSC thermal properties of debranched corn starch / chitosan / tannic acid complex

[0046] Example 2 Simulated in vitro gastrointestinal digestion experiments were conducted on different samples prepared in Example 1, as follows: Take 0.2 g of sample into an Erlenmeyer flask, add 25 mL of acetate-sodium acetate buffer (pH=5), take 500 μL of 0 h sample, add 300 μL of salivary amylase (prepared by adding 300 μL of α-amylase solution to 25 mL of pH 5.0 acetate-sodium acetate buffer), react for 5 min, add 15 mL of pepsin (prepared using 0.02 mol / L hydrochloric acid, enzyme concentration of 1 mg / mL), react at 37 ℃ in a shaker for 30 min, add 15 mL of 0.05 mol / L NaOH and 10 mL of compound enzyme (mix 120 μL of amylase and 20 μL of saccharifying enzyme, and adjust the volume to 10 mL using pH 5.0 acetate-sodium acetate buffer), react at 37 ℃, take 500 μL of sample after 20 min, 1 h and 2 h of reaction respectively, centrifuge at 10000 r / min for 10 min, take the supernatant and inactivate the enzyme in a boiling water bath for 10 min. After cooling, the glucose content in the reaction solution was determined using the grape oxidase method. The contents of rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) were calculated using the following formula: The glucose release process was fitted using a first-order kinetic model: ; In the formula: G 20 represents the glucose content released after 20 minutes, in mmol / L; G 120 represents the amount of glucose released after 120 minutes, in mmol / L. G 0 represents the free glucose content, mmol / L; 0.9 represents the conversion factor for the molar mass ratio of glucose to glucosinolate (a starch-forming unit). C t For the reaction t Cumulative glucose concentration at time point C ∞ This represents the theoretical endpoint concentration (mmol / L). k The digestion rate constant (min) -1 ).

[0047] During starch digestion, amylase first hydrolyzes the amorphous, non-crystalline regions of starch, gradually moving towards the crystalline regions. The in vitro digestion patterns of different samples are shown below. Figure 7As shown in Figure A, compared with WMS, the RS of DBS obtained after debranching treatment is significantly increased. This is because starch debranching produces more amylose, which enhances the starch's resistance to digestion. In the absence of CS, the addition of TA further increases RS and decreases RDS, attributed to TA's inhibition of α-amylase activity, slowing down the starch digestion rate. With only CS added, RS is slightly increased compared to DBS, while RDS further decreases, indicating that CS hinders enzyme-DBS contact to some extent through electrostatic adsorption and hydrogen bonding. Further addition of TA results in the highest RS content in DBS-1CS-TA, which is related to TA's inhibition of amylase activity and also reflects the synergistic effect of TA in enhancing the structural compactness of DBS-CS. However, in the three-phase system, further increasing the CS content leads to a decreasing trend in RS, while RDS correspondingly increases. This may be because excessive CS leads to a significant reduction in crystalline regions and an increase in amorphous structures, forming heterogeneous and partially loose regions that facilitate enzyme entry. This "over-crosslinking-crystalline region destruction" phenomenon ultimately weakens the antidigestion enhancement effect brought by TA.

[0048] Figure 7 The results from B show that the digestion curves of all treatments have a good fit with the first-order kinetic model, R0. 2 All values ​​were greater than 0.90, indicating that the model can reasonably describe the hydrolysis process of different complexes. Compared with other samples, DBS-1CS-TA had the smallest slope and the lowest corresponding hydrolysis rate constant k, indicating that its digestion rate was the slowest, consistent with the highest RS content shown in the in vitro digestion experiment. WMS had the highest slope, reflecting its fastest digestion, while the k values ​​of DBS, DBS-CS, and other three-phase complexes were all reduced, showing varying degrees of delayed digestion effects.

[0049] Example 3 1. Molecular docking Molecular docking was performed using Schrödinger 2025. The linear double helix starch structure was obtained from the Biotopics website (https: / / www.biotopics.co.uk) as the DBS. Four monosaccharide units were manually constructed in ChemDraw using β-1,4 bonds as the CS. The TA structure was obtained from PubChem (CID: 16129778). After importing all structures into Maestro, acceptor and ligand preparation was performed at pH 4.0 ± 2.0. Protonation state prediction, tautomer and stereoisomer generation, and partial charge distribution were performed using Epik. Energy minimization was then conducted using an OPLS force field.

[0050] The docking process consisted of two steps: First, an initial complex was constructed using a DBS double helix as the acceptor and CS as the ligand. The grid center was defined as (0, 0, -20), and the grid box size was set to 40×40×40 Å. Flexible docking was performed using the Ligand Docking module. The optimal DBS-CS conformation was selected based on the docking score. Then, the resulting complex was used as the acceptor, and a secondary docking was performed with TA as the ligand under the same grid parameters. The optimal DBS-CS-TA conformation was selected for analysis based on the docking score.

[0051] DBS consists of a stable double helix structure formed by two intertwined glucose chains, with numerous hydroxyl groups on its outer surface available for hydrogen bonding. Due to the large molecular weight of natural chitosan, which is unfavorable for docking and simulation, this invention uses a simplified fragment constructed from four β-1,4-linked glucosamine units to represent its typical structural features and ensure the feasibility and stability of the calculations. Under pH 4 conditions, acceptor and ligand treatment partially protonated the NH2 group of CS to -NH3. + Dating results ( Figure 8 The AC analysis shows that CS mainly adheres to the shallow surface region on the outer side of the DBS double helix. In the interface, the -NH3 group of CS... + Multiple hydroxyl groups form a dense network of hydrogen bonds with different glucose units of DBS, with bond lengths concentrated in the range of 1.8–2.5 Å. These hydrogen bonds are continuously distributed among multiple sugar units on both DBS chains, causing CS to exhibit a banded arrangement along the helical direction. The ΔG for MM-GBSA is -40.04 kcal / mol, indicating that the two form a stable binary complex.

[0052] After introducing TA ( Figure 8 In the ternary DF (dimethylformaldehyde) system, the binding mode changes. The TA polyphenol hydroxyl structure can form a wider range of multi-point hydrogen bonds on the DBS surface, while significantly expanding the interaction interface. Compared with CS, TA has more branches and occupies more space, forming a dense outer coating structure across different sugar chains of DBS. In addition, hydrogen bonds are also formed between TA and CS, enabling TA to "bridge" DBS and CS together, thereby constructing a synergistic network structure of the three. The MM-GBSA ΔG of the ternary system reaches -87.28 kcal / mol, corresponding to more stable binding.

[0053] 2. Molecular dynamics simulation The optimal conformations of DBS-CS and DBS-CS-TA obtained from docking were used as initial structures, and molecular dynamics simulations were performed using the Schrödinger-Desmond model. In System Builder, TIP3P was selected as the solvent model, and an Orthorhombic box was constructed, forming a simulation box of approximately 20 × 20 × 20 Å. The system used an OPLS4 force field, and appropriate ions were added to ensure electroneutrality. Simulations were performed for 100 ns in an NPT system at a temperature of 298.15 K and a pressure of 1 bar. One frame of trajectory was output every 100 ps, ​​and the obtained trajectories were used to calculate conformational stability indices such as RMSD and Rg.

[0054] Root mean square deviation (RMSD) is the most fundamental and commonly used structural characterization parameter in molecular dynamics simulations. It is used to quantitatively measure the conformational shift of a system over time, thus reflecting its overall stability and dynamic characteristics. RMSD can be used to assess the dynamic stability of a system during a 100 ns simulation. Figure 9 As shown in Figure AB, the RMSD curve exhibits a sharp upward trend in the initial stage of the simulation, reflecting the process by which the system rapidly approaches thermodynamic equilibrium from its initial conformation through structural relaxation, solvation, and intermolecular bonding adjustments. The curve then enters a relatively stable fluctuation phase, indicating that the system has reached dynamic equilibrium. The DBS-CS binary complex still exhibits significant oscillations during the equilibrium phase, while the RMSD fluctuation amplitude of the DBS-CS-TA ternary system after the introduction of TA is significantly reduced. This indicates that the addition of TA effectively restricts the excessive free movement of the DBS and CS skeletons, enhances the structural rigidity of the system, and is beneficial to improving the overall stability of the complex.

[0055] The radius of gyration (Rg) further reveals the spatial compactness and conformational characteristics of the complex. For example... Figure 9 As shown in CD, the Rg value of the DBS-CS complex remains at a high level, significantly higher than that of the single component, indicating that DBS and CS form a relatively relaxed, intertwined structure after binding. Consistent with the RMSD trend, the Rg curve of the DBS-CS binary system ( Figure 9 The C-type ternary system exhibits significant oscillations, indicating a certain degree of conformational variability; while the Rg curve of the DBS-CS-TA ternary system ( Figure 9 The middle (D) is significantly more convergent and smoother. This suppression of fluctuations further confirms the stabilizing role of TA in the system, which maintains a more compact and ordered spatial conformation of the complex by enhancing intermolecular interactions, effectively reducing the loosening and stretching of the skeleton.

[0056] In summary, this invention constructed a debranched corn starch-chitosan-tannic acid (DBS-CS-TA) ternary complex via self-assembly. Combined with multi-scale experimental characterization and molecular dynamics simulations, the structural assembly mechanism and in vitro digestion characteristics were elucidated. Results showed that the introduction of a small amount of CS could rearrange short chains on a larger scale through its template effect, resulting in an increase in relative crystallinity. Further addition of TA allowed its polyphenolic hydroxyl groups to compete with both DBS and CS for hydrogen bond sites, making the local structure more loosely structured. Simultaneously, its bridging effect enhanced the overall binding between chain segments, exhibiting a structural characteristic of overall compactness and local disorder. With increasing CS content, steric hindrance and multi-chain cross-linking made it difficult for chain segments to enter the crystalline region in an orderly manner, leading to a simultaneous decrease in crystallinity and short-range order. Molecular docking and MD simulations further supported this argument; both CS and TA could stably bind to the surface of DBS chain segments, and the multi-point hydrogen bond bridging of TA could reduce structural fluctuations in the system, making the ternary system more conformationally stable.

[0057] In in vitro digestion, the enzyme-inhibiting effect of TA, together with the physical barrier formed by CS-TA, significantly reduced the accessibility of amylase to starch chains, making the anti-digestion performance of the ternary complex system significantly better than that of single and binary systems. Among them, DBS-1CS-TA showed the best performance. Figure 10 The mechanism of action and anti-digestion resistance of the ternary system is summarized as follows: CS regulates the arrangement of DBS chain segments, TA acts as an interface bridging node, and the three together construct a multi-barrier structure, thereby achieving an overall improvement in anti-digestion resistance.

[0058] The ternary composite system constructed in this invention demonstrates the potential to effectively enhance the digestibility of starch through the synergistic effect of multiple components, providing a new feasible path for developing low-GI staple foods, sugar-controlled foods, and functional materials that stably deliver polyphenols. It also provides important inspiration for constructing starch-based composite systems with specific structure-digestion behavior.

[0059] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for improving the resistance to digestion of debranched corn starch, characterized in that, Includes the following steps: The debranched starch solution, chitosan solution, and tannic acid solution were mixed and reacted, and then centrifuged to obtain the precipitate. The precipitate was dried and ground to obtain a debranched starch-chitosan-tannic acid complex. The temperature of the mixing reaction is 4°C; The mixing reaction time is 24 hours; The pH value of the mixture reaction is 4.8~5.0; The concentration of the chitosan solution is 1-2 wt%; The debranched starch is obtained by enzymatic hydrolysis of gelatinized corn starch using pullulanase.

2. The method according to claim 1, characterized in that, The concentration of the chitosan solution is 1 wt%.

3. A debranched starch-chitosan-tannic acid complex prepared by the method according to any one of claims 1-2.

4. The application of the debranched starch-chitosan-tannic acid complex as described in claim 3 in the preparation of low-GI starch-based functional foods.

5. A low-GI starch-based functional food, characterized in that, Includes the debranched starch-chitosan-tannic acid complex as described in claim 3.

Images