Polyphenol-waxy highland barley starch compound as well as preparation method and application thereof

Abstract

The invention belongs to the field of food, and particularly relates to a polyphenol-waxy highland barley starch compound as well as a preparation method and application thereof. The preparation method of the polyphenol-waxy highland barley starch compound comprises the following steps: (1) mixing polyphenol and waxy highland barley starch to obtain a mixture; (2) dissolving the mixture into an ethanol solution, and stirring in a water bath to obtain a reactant; and (3) drying the precipitate obtained by centrifuging the reactant to obtain the polyphenol-waxy highland barley starch compound. According to the polyphenol-waxy highland barley starch compound, an in-vitro fermentation experiment shows that the polyphenol-waxy highland barley starch compound can be utilized by intestinal flora to generate short-chain fatty acid, and the composition of the intestinal flora is changed by promoting the growth of beneficial bacteria and reducing pathogenic bacteria, so that the intestinal health is maintained.

Description

Technical Field

[0001] This invention belongs to the food field, specifically relating to polyphenol-glutinous barley starch complex, its preparation method, and its application. Background Technology

[0002] Starch, as the main carbohydrate in grains, holds a central position in human nutrition and health. Its digestible properties (easily digestible starch RDS, slowly digestible starch SDS, and resistant starch RS) directly affect the prevention and control of diet-related diseases. Among them, resistant starch (RS), because it cannot be degraded by conventional digestive enzymes, plays a key role in blood sugar regulation, weight management, and gut microbiota balance, making it a focus of functional food research and development. Glutinous highland barley, a distinctive branch of highland barley, has extremely low amylose content in its grains and is rich in functional components such as β-glucan and total flavonoids. Its starch content reaches 50%–70%, with a significantly higher proportion of resistant starch than ordinary grains. Processed foods made from glutinous highland barley combine nutritional and health value with a unique taste, making it a high-quality raw material for meeting the health needs of specific populations and possessing significant development potential in the functional food field.

[0003] Despite the advantages of glutinous barley starch, such as its high resistant starch content, its in-depth development in functional foods is still limited by insufficient existing research. On the one hand, systematic studies on the structural characteristics, digestibility, and physiological functions of glutinous barley starch itself are lacking, failing to fully reveal its differentiating mechanism from ordinary starch. On the other hand, although polyphenols, as natural antioxidants, have been proven to alter the digestibility of starch by binding with it and possess bioactivities such as antioxidant, antibacterial, and gut microbiota regulation, the interaction mechanism between polyphenols and glutinous barley starch (such as structural binding mode and influence on starch physicochemical properties), the digestibility of the complex, and fermentation performance (especially its regulatory effect on gut microbiota composition) remain unclear. This makes it difficult to effectively utilize the synergistic health value of glutinous barley starch and polyphenols.

[0004] Given the current limitations in research on glutinous barley starch and the unclear mechanisms of action and functional properties of polyphenol-glutinous barley starch complexes, systematic research is urgently needed to fill these gaps. Clarifying the structural characteristics and interaction mechanisms between polyphenols (such as quercetin and its derivatives) and glutinous barley starch, and revealing their digestible properties and regulatory effects on gut microbiota during in vitro fermentation (such as promoting beneficial bacteria growth, inhibiting pathogenic bacteria, and producing short-chain fatty acids), will not only provide theoretical support for the high-value utilization of glutinous barley starch but also lay the foundation for developing novel functional foods with gut health benefits, thus playing a significant role in promoting the development of the nutrition and health food industry. Summary of the Invention

[0005] Based on this, the purpose of this invention is to provide a polyphenol-glutinous barley starch complex, its preparation method, and its application.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions: This invention provides a method for preparing a polyphenol-glutinous highland barley starch complex, the method comprising: (1) Mix polyphenols and glutinous barley starch to obtain a mixture; (2) Dissolve the mixture in an ethanol solution and stir in a water bath to obtain the reactants; (3) The precipitate after centrifugation of the reactants was dried to obtain polyphenol-glutinous barley starch complex.

[0007] Preferably, in the above preparation method, the polyphenols are selected from one or more of galangin, kaempferol, quercetin, or myricetin.

[0008] Preferably, in the above preparation method, the mass of polyphenols is 0.01% to 1% of the mixture; and / or the mass fraction of the ethanol solution is 45% to 55%.

[0009] Preferably, in the above preparation method, the temperature of the water bath is 55℃~65℃.

[0010] Preferably, in the above preparation method, the preparation method of glutinous highland barley starch includes: (a) Dry the glutinous highland barley, pulverize it, and sieve it to obtain glutinous highland barley powder; (b) Mix glutinous barley powder with an alkaline solution, stir, and centrifuge to obtain glutinous barley starch.

[0011] Preferably, in the above preparation method, the drying temperature is 40℃; and / or the sieving is done through a 100-mesh sieve; and / or the mass-to-volume ratio of glutinous highland barley powder to alkaline solution is 1g:10mL; and / or the centrifugation is done at 5000 r / min for 20 min.

[0012] In another aspect, the present invention provides a polyphenol-glutinous barley starch complex prepared by the above preparation method.

[0013] In another aspect, the present invention provides the application of the above-mentioned polyphenol-glutinous barley starch complex in maintaining intestinal health.

[0014] Preferably, in the above applications, maintaining gut health includes: promoting the proliferation of beneficial bacteria; and / or inhibiting the proliferation of pathogenic bacteria.

[0015] The beneficial effects of this invention include: through in vitro fermentation experiments, it can be seen that the polyphenol-glutinous barley starch complex provided by this invention can be utilized by intestinal flora to produce short-chain fatty acids, and can change the composition of intestinal microbiota by promoting the growth of beneficial bacteria and reducing pathogenic bacteria, thereby maintaining intestinal health. Attached Figure Description

[0016] Figure 1The scanning electron microscope (SEM) microstructures of different polyphenol-glutinous barley starch complexes are shown below. A1 is 1000x WHBS; A2 is 500x WHBS; B1 is 1000x GL-WHBS; B2 is 1000x KP-WHBS; C1 is 500x KP-WHBS; C1 is 1000x GL-WHBS; C2 is 500x KP-WHBS; D1 is 1000x QC-WHBS; D2 is 500x QC-WHBS. Figure 2 X-ray diffraction and crystallinity of different polyphenol-glutinous barley starch complexes; where (a) is WHBS; (b) is GL-WHBS; (c) is KP-WHBS; (d) is QC-WHBS; and (e) is MC-WHBS. Figure 3 Fourier transform infrared spectra of different polyphenol-glutinous barley starch complexes are shown; where (a) is WHBS; (b) is GL-WHBS; (c) is KP-WHBS; (d) is QC-WHBS; and (e) is MC-WHBS. Figure 4A The solubility of different polyphenol-glutinous barley starch complexes; Figure 4B The swelling power of different polyphenol-glutinous barley starch complexes; Figure 5 To assess the freeze-thaw stability of different polyphenol-glutinous barley starch complexes; Figure 6 A represents the in vitro digestibility characteristics of the galangin-glutinous highland barley starch complex; Figure 6 B represents the in vitro digestibility characteristics of the kaempferol-glutinous barley starch complex; Figure 6 C represents the in vitro digestibility characteristics of the myricetin-glutinous highland barley starch complex; Figure 6 D represents the in vitro digestibility characteristics of the quercetin-glutinous barley starch complex; Figure 7 A represents the quenching effect of different polyphenols on the α-amylase fluorophore; Figure 7 B represents the quenching inhibition rate of different polyphenols on the α-amylase fluorophore; Figure 8 The enzyme inhibition kinetics of α-amylase by different polyphenols; Figure 9 A represents the effect of different polyphenol-barley starch complexes on short-chain fatty acids (acetic acid); Figure 9 B represents the effect of different polyphenol-barley starch complexes on short-chain fatty acids (propionic acid); Figure 9 C represents the effect of different polyphenol-glutinous barley starch complexes on short-chain fatty acids (butyric acid); Figure 9 D represents the effect of different polyphenol-glutinous barley starch complexes on total short-chain fatty acids; Figure 10A The effects of different polyphenol-glutinous barley starch complex treatments on the phylum-level composition of the gut microbiota; Figure 10B The effects of different polyphenol-glutinous barley starch complex treatments on the composition of the gut microbiota at the genus level; Figure 10C Heatmap of the genus-level composition of gut microbiota after treatment with different polyphenol-glutinous barley starch complexes. Detailed Implementation

[0017] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0018] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0019] In a first aspect, embodiments of the present invention provide a method for preparing a polyphenol-glutinous highland barley starch complex, the preparation method comprising: (1) Mix polyphenols and glutinous barley starch to obtain a mixture; (2) Dissolve the mixture in an ethanol solution and stir in a water bath to obtain the reactants; (3) The precipitate after centrifugation of the reactants was dried to obtain polyphenol-glutinous barley starch complex.

[0020] It should be noted that this invention involves compounding glutinous barley starch with different polyphenols (such as galangin, kaempferol, quercetin, and myricetin) to form a polyphenol-glutinous barley starch complex. The glutinous barley starch exhibits a smooth, spherical surface with micropores. Compared to glutinous barley starch, the polyphenol-glutinous barley starch complex shows reduced interparticle spacing and enhanced particle aggregation. The polyphenols promote intermolecular interactions among glutinous barley starch particles, resulting in tighter packing and adhesion.

[0021] It should also be noted that, through analysis of the structure and physicochemical properties of low-GI glutinous barley starch, this invention shows that quercetin and its derivatives do not form inclusion complexes with starch. However, there is a significant linear relationship between the number of hydroxyl groups in phenolic compounds and the decrease in starch crystallinity. The addition of phenolic compounds alters the starch structure, solubility, and swelling capacity, thereby affecting its thermodynamic properties. Furthermore, this invention reveals that during the in vitro digestion of low-GI glutinous barley starch, the quercetin and its derivatives-glutinous barley starch complex spontaneously binds to α-amylase primarily through interaction with the hydroxyl groups of amino acid residues at the enzyme's active site, leading to significant inhibition of enzyme activity and hindering starch digestion. Moreover, there is a strong linear relationship between the number of hydroxyl groups in the phenolic compounds and the starch inhibition rate.

[0022] In some specific examples, in the above preparation method, the polyphenols are selected from one or more of galangin, kaempferol, quercetin, or myricetin.

[0023] In some specific examples, in the above preparation method, The polyphenols constitute 0.01% to 1% of the mixture, for example, 0.2%, 0.5%, or 0.7%; and / or The ethanol solution has a mass fraction of 45% to 55%, such as 47%, 50%, or 53%.

[0024] In some specific examples, the temperature of the water bath in the above preparation method is 55℃~65℃, such as 57℃, 60℃ or 62℃.

[0025] In some specific examples, the preparation method of glutinous highland barley starch in the above preparation method includes: (a) Dry the glutinous highland barley, pulverize it, and sieve it to obtain glutinous highland barley powder; (b) Mix glutinous barley powder with an alkaline solution, stir, and centrifuge to obtain glutinous barley starch.

[0026] In some specific examples, in the above preparation method, The drying temperature is 40℃; and / or Sieve through a 100-mesh sieve; and / or The mass-to-volume ratio of glutinous highland barley powder to alkaline solution is 1 g: 10 mL; and / or Centrifuge at 5000 r / min for 20 min.

[0027] Secondly, embodiments of the present invention provide a polyphenol-glutinous barley starch complex prepared by the above preparation method.

[0028] Thirdly, embodiments of the present invention provide an application of the above-mentioned polyphenol-glutinous barley starch complex in maintaining intestinal health.

[0029] In some specific examples, the above applications include maintaining gut health by promoting the growth of beneficial bacteria and / or inhibiting the growth of pathogenic bacteria.

[0030] It should be noted that this invention investigated the structural characteristics of four quercetin and its derivatives (such as galangin, kaempferol, quercetin, and myricetin)-glutinous barley starch (WHBS) complexes and their beneficial properties during in vitro digestion and fermentation. These polyphenols exhibited a binding capacity of >70% to WHBS, which increased with increasing concentration. Furthermore, due to the increased resistant starch moiety, the polyphenol-WHBS complexes inhibited starch digestion compared to gelatinized WHBS, and a significant linear relationship existed between the inhibition rate and the number of hydroxyl groups, in the order of galangin < kaempferol < quercetin < myricetin. The results showed that different complexes stimulated the growth of *Lactobacillus rhamnosus* and *Bifidobacterium* while inhibiting the growth of *Escherichia coli*. The inhibitory effect of quercetin and its derivatives on enzymes increased with increasing hydroxyl content in the structure. In addition, in vitro fermentation experiments showed that the complex was utilized by intestinal microorganisms to produce short-chain fatty acids. Furthermore, the polyphenol-WHBS complexes altered the composition of the intestinal microbiota by promoting the growth of beneficial bacteria and reducing pathogenic bacteria. The polyphenol-WHBS complex shows great potential as both a prebiotic and a probiotic, and has dual beneficial effects on the gut microbiota, providing a theoretical basis for the further development of polyphenol α-amylase inhibitors.

[0031] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0032] Preparation Examples Example 1 (1) Preparation of glutinous highland barley starch (1-1) Dry the glutinous barley in an oven (temperature 40℃), crush the dried sample and pass it through a 100-mesh sieve; (1-2) The sieved powder and sodium hydroxide solution (0.1 mol / L) were mixed with a magnetic stirrer at a certain ratio (the mass-volume ratio of powder to sodium hydroxide solution was 1:10 (g / mL)) and then centrifuged (5000 r / min for 20 min) to obtain the lower starch layer. (1-3) The lower starch layer was washed with ethanol (95% by mass) and deionized water in sequence, then filtered, dried in an oven at 45°C for 48 hours, pulverized, and passed through a 100-mesh sieve to obtain glutinous barley starch (WHBS).

[0033] (2) Preparation of polyphenol-glutinous barley starch complex (2-1) A mixture was obtained by mixing glutinous highland barley starch with galangin complex (GL), wherein the amount of galangin was 1% of the mass of the mixture; (2-2) Dissolve the mixture in 50% ethanol solution and stir in a water bath (temperature 60℃) to obtain the reactants; (2-3) The precipitate after centrifugation of the reactants was dried in an oven at 45°C for 48 hours and then ground to obtain the galangin-glutinous barley starch complex (GL-WHBS).

[0034] Example 2 Example 2 is largely the same as Example 1, except that Kaempferol complex (KP) is used in Example 2 to replace galangin in Example 1. Otherwise, the same as in Example 1 is used to prepare Kaempferol-WHBS (Starch-kaempferol complex).

[0035] Example 3 Example 3 is largely the same as Example 1, except that quercetin complex (QC) is used in Example 3 instead of galangin in Example 1. Otherwise, the same as in Example 1 is used to prepare quercetin-glutinous barley starch complex (QC-WHBS).

[0036] Example 4 Example 4 is largely the same as Example 1, except that in Example 4, myricetin (MC) is used instead of galangin in Example 1. Otherwise, the same as in Example 1 is used to prepare myricetin-glutinous barley starch complex (MC-WHBS).

[0037] Comparative Example 1 Comparative Example 1 is largely the same as Example 1, except that Comparative Example 1 does not use polyphenols to combine with glutinous barley starch. That is, Comparative Example 1 is the same as step (1) in Example 1, and glutinous barley starch (WHBS) is prepared.

[0038] Characterization test (1) Scanning electron microscopy microstructure The microstructure of the samples prepared in the examples and comparative examples was observed using a scanning electron microscope, as follows: The prepared samples were fixed on the sample stage with double-sided tape and gold-plated in a vacuum evaporator; the imaging conditions were 3.0 kV accelerating voltage, 8.9 mm working distance, and the surface shape of the samples was acquired at an appropriate magnification.

[0039] The results are as follows Figure 1 As shown, the results indicate that native glutinous barley starch (prepared in Comparative Example 1) has a smooth, round structure with some small pores; while the polyphenol-glutinous barley starch complex (prepared in the Example) has a rough surface and exhibits a polygonal structure with more pores. Due to the extremely low content of amylose in glutinous barley, the shrinkage of the starch is not obvious. Furthermore, the polyphenol-glutinous barley starch complex was prepared under mild heating conditions, which maintained the granular structure of the starch and promoted the absorption of polyphenols on the starch surface or their potential entry into the interior of the starch granules. Compared with native glutinous barley starch, the polyphenol-glutinous barley starch complex exhibits a more compact intergranular spacing, indicating that the addition of polyphenols causes the glutinous barley starch molecules to aggregate. This may be because polyphenols promote the interaction between starch granules, leading to increased particle aggregation and subsequent adhesion.

[0040] (2) X-ray diffraction and crystallinity test The crystallization properties of the samples prepared in the examples and comparative examples were tested using X-ray diffraction (XRD), specifically including: Cu-Kα radiation (λ=0.154 nm) at 40 kV and 40 mA, scanning from 5° to 60° (2θ) at a rate of 2° / min and an interval of 0.02°, and the scanning results were analyzed using MDI-jade 6.5 software.

[0041] The results are as follows Figure 2As shown, the results indicate that native glutinous barley starch (prepared in Comparative Example 1) and the polyphenol-glutinous barley starch complex (prepared in Example 1) have two strong reflection peaks near diffraction angles of 15° and 23°, and the diffraction peaks near 17° and 18° are connected double peaks, indicating that the crystal structure of glutinous barley starch is a typical type A. The characteristic peak at 19.8° is a marker of inclusion complex formation. Neither native starch nor the complex showed a significant characteristic peak at 19.8°, indicating that they did not form an inclusion complex. This may be because the content of amylose in glutinous barley is extremely low, which makes it difficult to form an inclusion complex. The double helix cavity formed by starch is extremely limited, while quercetin and its derivatives have relatively large molecular weights and significant steric hindrance, making it difficult for starch and polyphenols to form inclusion complex structures. Furthermore, the low solubility of polyphenols also hinders their interaction with starch. In addition, compared to native glutinous barley starch, the addition of polyphenols did not produce new crystallization peaks, indicating that there was no strong interaction between them to form special crystals. After the addition of polyphenols, the crystallinity of starch decreased from 20.01% to 18.47% (Starch-galangin complex), 18.02% (Starch-kaempferol complex), 17.23% (Starch-quercetin complex), and 16.82% (Starch-myricetin complex), respectively. The crystallinity decreased with increasing number of hydroxyl groups in the polyphenols. This is because hydroxyl groups not only reduce the binding force between starch molecules but also connect with starch through hydrogen bonds, hindering the formation of the amylose double helix structure, thus resulting in a regular decrease in crystallinity.

[0042] (3) Fourier transform infrared spectroscopy test The interactions and short-range ordered structures of the samples prepared in the test examples and comparative examples were determined using Fourier transform infrared spectroscopy, specifically including: using air as a reference, at a wavelength of 4000 cm⁻¹. -1 ~400cm -1 The area was scanned 64 times, with a resolution of 4cm. -1 For 1200cm -1 ~800cm -1 Deconvolve the feature peaks within the range (enhancement factor of 1.9, full width at half maximum of 20 cm). -1 ), calculate 1047cm -1 / 1022cm -1 The strength ratio.

[0043] The results are as follows Figure 3As shown, the results indicate that, compared to native glutinous barley starch (prepared in Comparative Example 1), the sample with added polyphenols (prepared in Example 1) did not exhibit peak formation or disappearance, indicating that the interaction between polyphenols and glutinous barley starch is non-covalent. Therefore, no new functional groups or covalent bonds were formed after addition, and the addition of polyphenols did not affect the molecular structure of starch; the FTIR spectrum at 3000 cm⁻¹... -1 ~3600cm -1 The spectral bands within this range are related to intermolecular hydrogen bonds. After the addition of polyphenols, the band at this point changes from 3409.63 cm⁻¹ of native starch. -1 They were moved to 3383.63cm respectively. -1 3379.55cm -1 3384.26cm -1 and 3386.33cm -1 At 2900 cm⁻¹, the absorption peak of glutinous barley starch shifts to lower wavenumbers, exhibiting a blue shift, indicating enhanced hydrogen bonding in the system. This is because both starch and polyphenols contain abundant hydroxyl groups, which can act as hydrogen acceptors and donors. They may interact through hydrogen bonds, thus improving the stability of the starch. -1 ~3000cm -1 The absorption peak at 1650 cm⁻¹ indicates the stretching vibration of CH. -1 The shear vibration caused by the two -OH groups of water molecules absorbed by the surface reflects the water molecules absorbed by the starch granules in the amorphous region. The peaks at both locations of glutinous barley starch did not change significantly after the addition of polyphenols, indicating that the addition did not affect the CH and moisture content in the amorphous region.

[0044] Additionally, 1047cm −1 and 1022cm −1 The bands at R1047cm are associated with the ordered and amorphous structures of starch, respectively. -1 / R1022cm -1 This is used to quantify the degree of order in starch samples; the calculation results are shown in Table 1. Compared with native starch, the R1047 cm⁻¹ of the polyphenol-starch complex is... -1 / R1022cm -1 All values ​​increased, and R1047cm increased with increasing number of hydroxyl groups. -1 / R1022cm -1 The increasing value indicates that the addition of hydroxyl groups may cause the crystal structure to shift towards amorphous regions, and the number of hydroxyl groups is positively correlated with the proportion of amorphous regions. This disrupts the formation of a relatively ordered structure and reduces crystallinity, which is consistent with the XRD results.

[0045] Table 1. Samples prepared in the Examples / Comparative Examples, R1047cm -1 / R1022cm -1

[0046] Performance testing (1) Solubility and swelling force test The following tests were conducted using the following methods for solubility and swelling power: 1 g (W0) of barley starch was accurately weighed into a centrifuge tube, 50 mL of water was added, and the mixture was shaken well. GL, KP, QC, and MC, each containing 5% (w / w) starch, were added separately. Centrifuge tubes containing only starch were used as the control group. The prepared sample solutions were incubated in a 90°C water bath for 30 min. After cooling to room temperature, the solutions were centrifuged at 4000 r / min for 20 min. The precipitate (P) was separated from the supernatant. The supernatant was poured into an aluminum box and dried in an oven at 50°C. The solution was weighed until a constant weight (W1) was achieved. The solubility and swelling power of the complex were calculated using the following formula: Solubility S% = W1 / W0 × 100; Swelling power E%= ; Where W0: sample mass, g; W1: sample supernatant mass after drying to constant weight, g; P: mass of precipitate after centrifugation, g.

[0047] Accurately weigh 0.6g of the sample (W0) prepared in the examples and comparative examples, add 30mL of deionized water to obtain a starch paste with a mass fraction of 2%, bath in a 95℃ water bath for 30min, centrifuge at 8000r / min for 20min; separate the supernatant and dry it in an oven at 105℃ to constant weight, weigh the mass of dissolved starch, decantate the precipitate and weigh it to obtain the mass of expanded starch (solubility and swelling power are important indicators of starch characteristics, and the solubility of starch is mainly related to the escaped amylose and amylopectin).

[0048] The solubility of the samples prepared in the examples and comparative examples is as follows: Figure 4A As shown, the results indicate that the addition of polyphenols increases solubility, with the order being galangin < kaempferol < quercetin < myricetin. This is because the hydroxyl groups on the polyphenols interact with the hydroxyl groups of starch molecules, promoting the swelling of starch granules by absorbing water. At the same time, due to the interaction between starch molecules and water molecules, starch exposes more groups that bind with water molecules, making the starch structure looser. Amylose and relatively smaller amylopectin molecules escape from the starch granules, thereby increasing the solubility of starch.

[0049] In addition, the expansion force of the samples prepared in the examples and comparative examples is as follows: Figure 4BAs shown, the results indicate that the addition of polyphenols reduces the swelling power of starch, and the degree of reduction is also in the order of galangin < kaempferol < quercetin < myricetin. This is because the hydroxyl groups on the polyphenols can bind to the outer chains of amylose and some amylopectin in glutinous barley through hydrophobic bonds, hydrogen bonds, and van der Waals forces, forming stable complexes with them. This restricts the expansion of glutinous barley starch during heating, reduces the specific surface area of ​​glutinous barley starch particles, and thus reduces the swelling power of glutinous barley starch.

[0050] (2) Freeze-thaw stability test Accurately weigh 0.9g of the sample (W0) prepared in the examples and comparative examples, and add 30mL of deionized water to obtain a starch paste with a mass fraction of 3%. Incubate in a 95℃ water bath for 30min, and then cool to room temperature. Then weigh the centrifuge tube, add 30.00g of starch paste to the centrifuge tube, weigh the total mass of the centrifuge tube and starch paste, and then freeze in a refrigerator for 24h. Thaw naturally, centrifuge at 5000 r / min for 20min, and weigh the total mass of the centrifuge tube and precipitate to test the freeze-thaw stability (the stability of starch during freeze-thaw is an important indicator of the quality maintenance of starchy foods during freezing, and can be determined by the water separation rate after thawing of starch; the higher the water separation rate, the worse the freeze-thaw stability of starch).

[0051] The results are as follows Figure 5 As shown, the results indicate that the addition of polyphenols reduces the water separation rate of glutinous barley starch, thus improving its freeze-thaw stability. The more hydroxyl groups there are, the higher the freeze-thaw stability, which makes it easier for the polyphenols to interact with amylose through hydrogen bonds, thereby altering the intermolecular forces of glutinous barley starch, promoting the formation of a spongy starch gel network, and reducing gel separation.

[0052] (3) In vitro digestion characteristics test Accurately weigh 0.9g of the sample (WO) prepared in the examples and comparative examples, add 30mL of deionized water to prepare a starch paste with a mass fraction of 3%, incubate at 95℃ for 20min, cool to room temperature, dry in an oven to constant weight, grind, and pass through a 100-mesh sieve to obtain the sample; dissolve 0.2g of the sample in 15mL of phosphate buffer (pH=5.8), incubate at 37℃ for 5min, add 5mL of mixed enzyme solution (α-amylase 290u / mL, saccharifying enzyme 15u / mL), shake in a 37℃ water bath, and hydrolyze for 0, 20min and 120min respectively. Take 1mL of the hydrolysate, add 8mL of anhydrous ethanol to inactivate, centrifuge (5000r / min, 10min), and determine the reducing sugar content in the supernatant by DNS colorimetric method.

[0053] Test results are as follows Figure 6 A to Figure 6As shown in Figure D, the results indicate that the RDS content of native glutinous barley starch (prepared in Comparative Example 1) was 67.29±3.36%, the SDS content was 14.22±0.71%, and the RS content was 18.49±0.92%. The RDS content of glutinous barley starch with added polyphenols (prepared in the Example) decreased slightly, while the SDS and RS contents increased slightly. With the increase of the polyphenol addition ratio, the RDS content of glutinous barley starch gradually decreased, while the RS content gradually increased. Myricetin, which has the most significant anti-digestion effect, showed that when the mixing ratio reached 1%, the RDS content decreased from 67.29±3.36% to 56.01±2.82%, while the RS content increased from 18.49±0.92% to 26.22±1.29%.

[0054] As mentioned above, the hydrolysis rate of starch is closely related to many factors, including particle size, surface structure, the ratio of amylose to amylopectin, and crystal type. The hydrolysis rate of starch gradually decreases with the addition of polyphenols, and the concentration of polyphenols is positively correlated. Furthermore, it can be seen that the resistance to digestion is in the order of galangin < kaempferol < quercetin < myricetin. In addition, although the resistance of polyphenols to starch digestion is positively correlated with the polyphenol concentration before a mixing ratio of 1%, in preliminary experiments, the resistance to digestion decreased after the mixing ratio exceeded 1%. This is because the dextrins and disaccharides produced by acid hydrolysis of starch may be easily digested by enzymes. The acidic conditions induced by polyphenols can stably increase the starch hydrolysis rate. When excessive polyphenols are added, the starch structure may become more loose, thus providing more opportunities for digestive enzymes to hydrolyze the starch.

[0055] (4) Test of the binding strength between polyphenols and glutinous highland barley starch Glutinous barley starch was mixed with galangin, kaempferol, quercetin, and myricetin at a mass ratio of 99:1 (glutinous barley starch: polyphenol) to obtain mixed powders. 3 mg of the mixed powder and 9 μL of distilled water were added to an aluminum crucible, while 3 mg of single glutinous barley starch powder was used as a control. All samples were stored at 4 °C overnight and then heated from 30 °C to 130 °C at a rate of 10 °C / min. The onset temperature (To), peak temperature (Tp), end temperature (Tc), and enthalpy change (ΔH) were measured, and the results are shown in Table 2 below.

[0056] Table 2. Effects of polyphenols on the gelatinization heat properties of glutinous barley starch

[0057] Table 2 shows that the initial temperature (To) of starch was not significantly affected by the addition of polyphenols, but the peak temperature (Tp), termination temperature (Tc), and gelatinization enthalpy (ΔH) all decreased, indicating that the energy required for gelatinization of glutinous barley starch was reduced after polyphenol adsorption. This is because the hydrophilic hydroxyl groups in polyphenols alter the gelatinization heat characteristics of starch by reducing the interaction between starch and water molecules. Simultaneously, the hydroxyl groups combine with the amorphous regions of starch granules, changing the binding force between the crystalline and amorphous regions, thus lowering the gelatinization enthalpy, which confirms the XRD results.

[0058] (4) Fluorescence quenching test The fluorescence quenching spectra of GL, KP, QC, and MC on amylase (α-amylase) were measured using a fluorometer (Shimadzu XRF 1800, Japan); details are as follows: (4-1) First, dissolve porcine pancreatic α-amylase at 33.75 U / mL in phosphate buffer (0.2 M, pH 6.8).

[0059] Different polyphenol concentrations (i.e., 0.05, 0.1, 0.2, 0.4, ...) were prepared in phosphate buffer (0.2 M, pH 6.8). 0.8 mg / ml).

[0060] (4-2) Then, mix GL, KP, QC and MC (0.2 mL) with enzyme solution (3 mL) by vortexing for 3 min, and then make up to 10 mL to obtain a mixture; (4-3) The mixture was incubated in constant temperature water baths at 30°C and 37°C for 30 min respectively; the control group used phosphate buffer containing 3 mL of enzyme solution; the fluorescence emission spectrum was scanned in the wavelength range of 290 nm to 500 nm, i.e. (excitation wavelength: 278 nm, emission wavelength: 290 nm, slit width: 5 nm).

[0061] Test results are as follows Figure 7 A and Figure 7As shown in Figure B, the results indicate that the Stern-Volmer curves of the interaction between phenolic compounds and α-amylase at two different temperatures (30°C and 37°C) exhibit a strong linear relationship. At both temperatures, the slope of the curves is ordered as follows: galangin < kaempferol < quercetin < myricetin, indicating that the more hydroxyl groups present, the more significant the quenching effect of polyphenols on the α-amylase fluorophore, i.e., the stronger the binding strength with amylase. Furthermore, the Ka value is close to 10³ L / (mol·s), and the n value is close to 1, indicating that their binding strength with α-amylase is moderate, and they have only one or a class of binding sites on the amylase. Kq increases with increasing temperature but is much higher than the maximum dynamic quenching constant, indicating that the fluorescence quenching process of quercetin and its derivatives on α-amylase is mainly static quenching, supplemented by dynamic quenching.

[0062] In addition, as shown in Table 3, the ΔG values ​​of quercetin and its derivatives are all negative, indicating that their binding to α-amylase is spontaneous; furthermore, the ΔH>0 and ΔS>0 values ​​of the four polyphenols indicate that the main interaction between them and α-amylase is a hydrophobic interaction.

[0063] Table 3. Fluorescence quenching binding and thermodynamic parameters of the interaction between α-amylase and phenolic compounds at different temperatures.

[0064] (5) Enzyme inhibition kinetics test of α-amylase The α-amylase inhibition patterns of different polyphenols were determined using the Michaelis-Menten and Lineweaver-Burk models; inhibition kinetics were studied using a similar method described in the α-amylase activity assay, with the difference being that the final polyphenol concentrations were 0.4 mg / mL and 0.6 mg / mL, and the starch solution concentrations were 0.5%, 1%, 1.5%, and 2% (w / v), respectively; one tube of each concentration was taken every 5 minutes and 1.0 mL of DNS reagent was added; the reaction mixture was heated in boiling water for 5 minutes, then cooled to room temperature, and then brought to a constant volume of 25 mL; the absorbance at 540 nm was measured using a UV-Vis spectrophotometer.

[0065] Test results are as follows Figure 8As shown, the results indicate that the inhibitory effect of quercetin and its derivatives on the enzyme increases with the increase of hydroxyl content in their structure, in the order of galangin < kaempferol < quercetin < myricetin, and the effect increases with increasing concentration. Myricetin, at a concentration of 1 mg / mL, exhibited an excellent inhibitory effect on α-amylase, reaching an inhibition rate of 30.97 ± 1.42%. This order is consistent with their anti-digestion rate; more hydroxyl groups result in better inhibitory effects. This suggests that quercetin and its derivatives mainly inhibit starch digestion by interacting with amino acid residues at the enzyme's active site through hydroxyl groups, thereby reducing α-amylase activity.

[0066] (6) Test on the effect of polyphenol-starch complex on short-chain fatty acids In vitro digestion experiments showed that the addition of four polyphenols decreased the content of readily digestible starch (RDS) in WHBS, while increasing the content of resistant starch (RS). Furthermore, RS levels correlated with hydroxyl content. It is well known that resistant starch produces various beneficial short-chain fatty acids (SCFAs) during colonic fermentation, thus maintaining gut microbiota balance. However, how these different polyphenolic complexes affect the gut microbiota during in vitro fermentation remains unclear. Acetic acid, one of the fermentation products of most bacteria, can directly act on hypothalamic mechanisms to enhance satiety and suppress appetite. Based on this, the concentration changes of acetic acid, propionic acid, and butyric acid in the samples prepared in the examples and comparative examples were tested after fermentation for 0-24 hours. The conditions used were as follows: N2, 25 mL / min; air flow rate, 300 mL / min; H2 flow rate, 30 mL / min; inlet temperature, 250 °C; detector temperature, 280 °C; the temperature was programmed, with the initial temperature of the column set at 80 °C for 2 minutes, then heated to the final temperature of 180 °C at a rate of 6 °C / min and held for 4 minutes; and a sample injection volume of 1 μL was used.

[0067] Test results are as follows Figure 9 A to Figure 9 As shown in Figure D, the results indicate that after 24 hours of fermentation, the concentrations of the three SCFAs in the four polyphenol-glutinous barley starch complexes significantly increased; polyphenols may reshape the microbial community composition by stimulating the proliferation of specific short-chain fatty acid-producing microorganisms. Specifically, as... Figure 9As shown in Figure A, after 12 hours of fermentation, the acetic acid content in the polyphenol-added groups was significantly higher than that in the control group. Among them, GL, KP, QC, and MC increased the acetic acid concentration from 492.75 μg / mL to 516.28 μg / mL, 503.96 μg / mL, 538.11 μg / mL, and 556.45 μg / mL, respectively. After 24 hours of fermentation, the acetic acid yield increased to 730.1 μg / mL, 757.08 μg / mL, 818.13 μg / mL, and 891.25 μg / mL, respectively; the total acid yield reached 952.22 μg / mL, 973.07 μg / mL, 1001.66 μg / mL, and 1136.80 μg / mL, all of which were significantly higher than those in the control group without polyphenols. As can be seen from the above, the addition of MC results in significantly higher levels of propionic acid, butyric acid, and total acid compared to other ingredients. This regulatory effect can more efficiently promote the conversion of substrates such as fiber and starch into short-chain fatty acids by intestinal microorganisms. Resistant starch can be fermented by intestinal flora to produce butyric acid, and the presence of polyphenols may further enhance this process—especially when they coexist with fermentable dietary fibers such as β-glucan, which can significantly increase the levels of propionic acid and butyric acid.

[0068] In summary, the synergistic effect of polyphenols and starch promotes the production of acetic acid, propionic acid, and butyric acid by regulating the microbial community, slowing down the digestion process, improving substrate utilization, and modulating fermentation metabolic pathways; the higher the hydroxyl content, the higher the conversion rate of short-chain fatty acids, which is in complete agreement with previous in vitro experimental results.

[0069] (7) Test on the effect of polyphenol-glutinous barley starch complex on gut microbiota This test recruited four adult volunteers with no recent history of gastrointestinal disease or any history of use of probiotics, prebiotics or antibiotics, and fresh stool samples were obtained from them; the fresh stool samples were suspended (10% w / v) in sterile phosphate buffer and homogenized to obtain stool homogenate; the stool homogenate was filtered through clean gauze to obtain stool slurry, which was then used for fermentation; the composition of the growth medium is listed in Table 4; Table 4 Composition of growth medium

[0070] The following six groups were used for fermentation, which lasted for 24 hours: no carbon source (blank, blank control), galactooligosaccharides (P-control, positive control), starch with the same treatment (control, control), and WHBS, or GL-WHBS, KP-WHBS, QC-WHBS, and MC-WHBS; the fermentation broth in each flask (6 ml) consisted of growth medium (5 ml), fecal homogenate (1 ml), and each of the above carbon sources (48 mg each); the fermentation flasks were transferred to an anaerobic incubator maintained at 37°C, and fermentation was collected at 0, 12, and 24 hours. Liquid was used for further analysis (samples were in separate flasks set up for each duration); experimental setups included three replicates per group and at each time point; fermentation samples were collected 24 hours after fermentation for analysis of microbial quality and composition; genomic DNA was extracted using the CTAB / SDS method and subsequently analyzed by Mingke Biotechnology Co., Ltd.; high-throughput sequencing of the 16S rDNA V4 region was performed using the Illumina Miseq platform; microbial complexity and intergroup differences were investigated using high-quality, clean data based on operational taxonomic units and annotation results.

[0071] Further analysis and evaluation of the composition and abundance of the gut microbiota produced by short-chain fatty acids (SCFAs) revealed significant changes in gut microbiota composition after 24 hours of fermentation, as shown in the following results. Figure 10A As shown, the results indicated that Bacteroidetes, Actinobacteria, and Proteobacteria were the most abundant phyla. Among them, the relative abundance of Actinobacteria increased in the groups treated with the four polyphenol-glutinous barley starch complexes compared to the control group; additionally, the abundance of Bacteroidetes increased, including many SCFA-producing strains, particularly butyrate-producing bacteria, which are associated with enhanced gut health. Changes in Bacteroidetes abundance directly reflect efficient carbohydrate utilization, indicating a prebiotic effect. Actinobacteria include the probiotic genus Bifidobacterium, which ferments carbohydrates to produce lactic acid and acetic acid, inhibiting pathogens, enhancing immunity, and promoting the colonization of beneficial bacteria. These beneficial genera showed a significant increase compared to the polyphenol-free control group. p <0.05), indicating that polyphenol supplementation effectively regulates the gut microbiota and enriches the probiotic population.

[0072] In addition, such as Figure 10B As shown, the dominant taxa included Bifidobacteria and Bacteroides. The polyphenol-barley starch complex exhibited varying degrees of probiotic effects compared to the control group, particularly a significant increase in the relative abundance of Bifidobacterium adolescenti, a species known to play a role in carbohydrate fermentation. This finding is consistent with previously observed increases in acetic acid and total acid production.

[0073] also, Figure 10CA heatmap of gut microbiota composition at the genus level was presented, identifying 35 distinct genera in human fecal samples. The following genera were predominant: Bacteroides, Parabacteroides, Enterococcus faecalis, Bifidobacterium, Ruminococcus, Anaerobic Bacteria, Parabacteroides, Roseobacterium, Bacteroides fulminatus, Streptococcus, and Broutella; In the presence of all polyphenol-glutinous barley starch complexes, compared to the control group, Parasutterella and Sutterella Growth is inhibited.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a polyphenol-glutinous barley starch complex, characterized in that, Preparation methods include: (1) Mix polyphenols and glutinous barley starch to obtain a mixture; (2) Dissolve the mixture in an ethanol solution and stir in a water bath to obtain the reactants; (3) The precipitate after centrifugation of the reactants was dried to obtain polyphenol-glutinous barley starch complex.

2. The preparation method according to claim 1, characterized in that, The polyphenols are selected from one or more of galangin, kaempferol, quercetin, or myricetin.

3. The preparation method according to claim 1 or 2, characterized in that, The mass of polyphenols is 0.01% to 1% of the mixture; and / or The ethanol solution has a mass fraction of 45% to 55%.

4. The preparation method according to claim 1 or 2, characterized in that, The water bath temperature is 55℃~65℃.

5. The preparation method according to claim 1 or 2, characterized in that, The preparation methods of glutinous highland barley starch include: (a) Dry the glutinous highland barley, pulverize it, and sieve it to obtain glutinous highland barley powder; (b) Mix glutinous barley powder with an alkaline solution, stir, and centrifuge to obtain glutinous barley starch.

6. The preparation method according to claim 5, characterized in that, The drying temperature is 40℃; and / or Sieve through a 100-mesh sieve; and / or The mass-to-volume ratio of glutinous highland barley powder to alkaline solution is 1 g: 10 mL; and / or Centrifuge at 5000 r / min for 20 min.

7. A polyphenol-glutinous barley starch complex prepared by the preparation method according to any one of claims 1 to 6.

8. The use of the polyphenol-glutinous barley starch complex according to claim 7 in maintaining intestinal health.

9. The application according to claim 8, characterized in that, Maintaining gut health includes: Promote the growth of beneficial bacteria; and / or Inhibit the proliferation of pathogenic bacteria.

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