Dietary fiber of kiwi peel residue with different combined polyphenol release degree, preparation method and application

Abstract

This invention discloses a dietary fiber from kiwifruit peel residue with different levels of polyphenol release, its preparation method, and its applications. Using kiwifruit peel residue as raw material, the starch and protein are removed by enzymatic hydrolysis to obtain dietary fiber. Then, alkaline hydrolysis and combined alkaline hydrolysis with ultrasonic treatment are used to prepare kiwifruit peel residue dietary fiber samples with different levels of polyphenol release. The release amount of polyphenols, changes in antioxidant activity, intestinal flora regulation, and short-chain fatty acid production capacity of each sample were evaluated. The A8MUS sample significantly increased the relative abundance of beneficial bacteria such as Bacteroides, decreased the relative abundance of conditionally pathogenic bacteria such as Escherichia coli and Providencia, and promoted the production of short-chain fatty acids such as acetic acid and isobutyric acid. This invention realizes the high-value utilization of kiwifruit peel residue by-products, and the obtained dietary fiber can be used as a functional food ingredient or an intestinal microecological regulator.

Description

Technical Field

[0001] This invention relates to the field of agricultural by-product processing technology, specifically to a dietary fiber from kiwifruit peel residue with different polyphenol release levels, its preparation method, and its application. Background Technology

[0002] Kiwifruit is rich in vitamins, polyphenols, and dietary fiber, offering significant nutritional and health benefits. However, kiwifruit processing (such as juice and jam production) generates numerous byproducts. The peel and pomace are typically treated as waste, leading to resource waste and environmental burden. Research indicates that kiwifruit peels and pomace still contain abundant dietary fiber and bound phenolic compounds, which possess various bioactivities, including antioxidant, anti-inflammatory, and gut microbiota-regulating properties. Therefore, the high-value utilization of kiwifruit peels and pomace to develop functional dietary fiber products holds significant economic and environmental importance.

[0003] Phenolic compounds can be classified into free phenols and bound phenols based on their degree of binding with dietary fiber. Bound phenols are tightly bound to the polysaccharide chains of dietary fiber through ester bonds, ether bonds, or hydrogen bonds, making them difficult to release under normal digestion conditions and limiting their bioavailability in vivo. How to disrupt the dense structure of dietary fiber and weaken its binding with phenols through appropriate physical or chemical treatments, thereby increasing the release rate of bound phenols, has become a current research hotspot.

[0004] In existing technologies, alkaline hydrolysis is a common method for disrupting cell wall structure and releasing bound phenols. However, alkaline treatment alone is often insufficient to fully break down the highly cross-linked structure of dietary fiber. Ultrasonic-assisted extraction technology utilizes cavitation effects to further break down the fiber network and enhance the dissolution of active ingredients. However, systematic studies on the release patterns of bound phenols, changes in antioxidant activity, and regulatory effects on gut microbiota during simulated gastrointestinal digestion and colonic fermentation of dietary fibers with varying degrees of bound polyphenol release have not yet been adequately reported. Furthermore, the effects of different treatment conditions on the microstructure of dietary fiber and its ability to generate short-chain fatty acids require further clarification.

[0005] Therefore, developing a preparation method that can effectively regulate the binding degree of dietary fiber in kiwifruit peel residue, increase the release of bound phenols, and enhance intestinal probiotic functions is of great significance for the high-value utilization of kiwifruit processing by-products and the development of functional foods. Summary of the Invention

[0006] To address the shortcomings of the existing technologies, this invention aims to provide a dietary fiber from kiwifruit peel residue with varying degrees of polyphenol release, its preparation method, and its application, thereby realizing the utilization of kiwifruit peel residue by-products.

[0007] To solve the above problems, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing dietary fiber from kiwifruit peel and pomace with different degrees of polyphenol release, comprising the following steps: Kiwifruit peel residue freeze-dried powder was subjected to high-temperature α-amylase, alkaline protease and saccharifying enzyme treatment to remove starch and protein, and then washed with ethanol, washed with distilled water and freeze-dried to obtain kiwifruit peel residue dietary fiber KPDF. KPDF was mixed with NaOH solutions of different concentrations at a ratio of 1:30 (w / v), and the mixture was sealed in a container and hydrolyzed by shaking at room temperature to obtain kiwifruit peel residue dietary fiber with different degrees of polyphenol release.

[0008] Furthermore, the concentration of the NaOH solution is 2-10M, and the content of dietary fiber-releasing bound phenols is 5.94-15.57 mg GAE / g.

[0009] Furthermore, the NaOH solution concentration is 8M, and the dietary fiber releases bound phenols with a content of 15.57±0.19 mgGAE / g.

[0010] Furthermore, after hydrolysis in an 8M NaOH solution, the dietary fiber was subjected to ultrasonic treatment for 0-75 minutes, resulting in the release of 14.86-22.52 mg GAE / g of bound phenols from the dietary fiber.

[0011] Furthermore, after hydrolysis in an 8M NaOH solution, the dietary fiber was subjected to ultrasonic treatment for 45 minutes, resulting in a phenol content of 22.52 ± 0.45 mg GAE / g.

[0012] Secondly, the present invention provides a kiwifruit peel residue dietary fiber with different polyphenol release levels prepared by the method described above.

[0013] Furthermore, the dietary fiber in kiwifruit peel and pulp has a loose and porous microstructure; The total phenol release from the dietary fiber bound in kiwifruit peel and pulp reached 13.29±0.09 mgGAE / gDW and 27.4±0.46 mgGAE / gDW 24 hours after entering the small intestine digestion stage and the colon fermentation stage, respectively.

[0014] Thirdly, the present invention provides the application of the kiwifruit peel and pomace dietary fiber with different levels of bound polyphenol release in the preparation of products for regulating intestinal flora, wherein the regulation of intestinal flora includes at least one of the following effects: Increase the α and β diversity of fecal microbiota; Reduce the relative abundance of Proteobacteria; Increase the relative abundance of Bacteroides; Reduce the relative abundance of opportunistic pathogens such as Shigella and / or Providencia.

[0015] Fourthly, the present invention provides the application of the kiwifruit peel and pomace dietary fiber with different polyphenol release levels in the preparation of products for promoting the generation of short-chain fatty acids, wherein the short-chain fatty acids include acetic acid and / or isobutyric acid.

[0016] Furthermore, the product is a food or an intestinal microecological regulator.

[0017] The beneficial effects of this invention are as follows: (1) By using 8M NaOH combined with ultrasonic treatment for 45 min, the dense structure of dietary fiber can be significantly destroyed, the total phenol release reaches 27.4 mg GAE / gDW, and the antioxidant activity is significantly improved.

[0018] (2) After 24 hours of fermentation in the colon, the dietary fiber obtained binds to phenol release at its peak and can effectively regulate the intestinal flora: increase the relative abundance of beneficial bacteria such as Bacteroides and decrease the relative abundance of conditionally pathogenic bacteria such as Shigella.

[0019] (3) Promotes the production of short-chain fatty acids (acetic acid, isobutyric acid) and improves the intestinal microecological environment.

[0020] (4) To realize the high-value utilization of kiwifruit peel residue by-products, which can be used as functional food ingredients or intestinal microecological regulators. Attached Figure Description

[0021] Figure 1 The effect of different NaOH concentrations on the total phenol content of KPDF.

[0022] Figure 2 The effect of different NaOH concentrations on the antioxidant activity of KPDF extract.

[0023] Figure 3 The effect of ultrasonic time on the release of total phenols in KPDF under the condition of NaOH concentration of 8M.

[0024] Figure 4 To investigate the effect of ultrasonic time on the antioxidant activity of KPDF extract under NaOH concentration of 8M.

[0025] Figure 5 This is a SEM image of dietary fiber from KPDF.

[0026] Figure 6 This is a SEM image of A2M dietary fiber.

[0027] Figure 7 This is a SEM image of A8M dietary fiber.

[0028] Figure 8This is a SEM image of dietary fiber from an A8MUS sensor.

[0029] Figure 9 The total phenol content of dietary fiber with different levels of polyphenol release during simulated gastrointestinal digestion and colonic fermentation processes.

[0030] Figure 10 To evaluate the changes in antioxidant activity of dietary fiber with different polyphenol release levels during in vitro simulated gastrointestinal digestion and colonic fermentation using the DPPH method.

[0031] Figure 11 To evaluate the changes in antioxidant activity of dietary fiber with different polyphenol release levels during in vitro simulated gastrointestinal digestion and colonic fermentation using the ABTS method.

[0032] Figure 12 To evaluate the changes in antioxidant activity of dietary fiber with different polyphenol release levels during in vitro simulated gastrointestinal and colonic fermentation processes using the FRAP method.

[0033] Figure 13 The fecal microbiota α diversity was evaluated by the Shannon and Simpson indices for dietary fiber with different levels of polyphenol release.

[0034] Figure 14 To evaluate fecal microbiota β diversity using PCoA analysis.

[0035] Figure 15 The relative abundance of bacterial communities at the phylum level.

[0036] Figure 16 The relative abundance of genus-level bacterial communities.

[0037] Figure 17 The relative abundance of Bacteroides is shown.

[0038] Figure 18 The relative abundance of Escherichia coli and Shigella spp.

[0039] Figure 19 The relative abundance of the genus Providencia. Detailed Implementation

[0040] The present invention will be further described in detail below with reference to specific embodiments.

[0041] It should be noted that these embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Simple improvements to the method under the premise of the present invention are all within the scope of protection claimed by the present invention.

[0042] Example 1 The kiwifruit variety used was Xuxiang kiwifruit, produced in Meixian County, Shaanxi Province. Fresh peel and pulp were stored at -20℃, freeze-dried, and then passed through an 80-mesh sieve to obtain kiwifruit peel and pulp freeze-dried powder (KP). Alkaline protease was purchased from Novozymes. High-temperature α-amylase, saccharifying enzyme, Folin-Ciocalteu, ABTS, DPPH, pepsin, trypsin, and pig bile powder were purchased from Yuanye Biotechnology Co., Ltd. Acetic acid, propionic acid, butyric acid, isobutyric acid, isovaleric acid, valeric acid, and diethylbutyric acid were purchased from Maclean Biochemical Technology Co., Ltd., and all were GC grade.

[0043] 1.1 Preparation of kiwifruit peel and pulp dietary fiber (KPDF) 50g KP was mixed with 500mL PB buffer, and the pH of the solution was adjusted to 6. 2mL of high-temperature α-amylase was added, and the mixture was incubated at 40℃ for 1.5h. Then, the pH was adjusted to 7.5, and 2.5mL of alkaline protease was added, and the mixture was incubated at 40℃ for 1h. Next, the pH was adjusted to 4.5, and 1mL of saccharifying enzyme was added, and the mixture was incubated at 40℃ for 1h to remove impurities such as starch and protein. The mixture was then centrifuged (8000 rpm, 4℃, 10 min) to collect the precipitate. The precipitate was washed twice with 80% ethanol at a 1:10 (w / v) ratio to remove free phenols. The mixture was repeatedly washed with distilled water, freeze-dried for 48h, and the resulting dietary fiber sample was pulverized and passed through an 80-mesh sieve to obtain KPDF, which was stored at -20℃ for later use.

[0044] 1.2 Preparation of dietary fiber with different degrees of binding and extraction of its phenolic substances Dietary fiber with different binding degrees was prepared by alkaline hydrolysis and alkaline hydrolysis combined with ultrasonication. 1 g KPDF was mixed with different concentrations of NaOH solution (2M, 4M, 8M, 10M) at a ratio of 1:30 (w / v). Hydrolysis was carried out at room temperature with shaking at 180 rpm for 4 h. Based on the determined NaOH concentration, the ultrasonic conditions were further optimized: 8M NaOH was preferred for treatment followed by ultrasonication (300 W, 40℃), with controlled ultrasonication times (0 min, 15 min, 30 min, 45 min, 60 min, 75 min). Subsequently, the pH of the solution was adjusted to 1-3, and the mixture was centrifuged (4℃, 10000 rpm, 10 min). The precipitate was washed twice with pure water to obtain dietary fiber with different binding degrees. The supernatant was extracted five times with an equal volume of ethyl acetate. The ethyl acetate organic phases were combined and evaporated to dryness at 45°C. The volume was then adjusted to 10 mL with methanol to obtain phenols with different release levels, which were used to determine the content of phenolic substances and antioxidant activity. Several dietary fibers with different binding levels were selected. The corresponding precipitates were thoroughly mixed with pure water at a ratio of 1:30 (w / v), centrifuged (10000 r, 4°C, 10 min), and this operation was repeated twice. The resulting precipitates were then freeze-dried for the characterization of the microstructure of dietary fibers.

[0045] 1.3 Content of phenolic substances and their antioxidant activity 1.3.1 Determination of total phenol content The total phenolic content of bound polyphenols in kiwifruit peel and pomace dietary fiber (KPDF) was determined using the Folin-Ciocalteu method. The procedure was as follows: 1 mL of sample solution was taken, and 0.5 mL of 50% Folin-Ciocalteu reagent was added; subsequently, 1 mL of 5% sodium carbonate solution was added, and the mixture was shaken and mixed for 1 min. The mixture was then incubated at 25℃ in the dark for 60 min, and the absorbance was measured at 760 nm. The total phenolic content is expressed as mgGAE / gDW.

[0046] 1.3.2 Antioxidant Activity Assay 1.3.2.1 DPPH free radical scavenging ability The DPPH radical scavenging activity of the samples was determined by referring to existing literature methods with slight modifications. 200 μL of sample solution was taken and 1.8 mL of 0.2 mmol / L DPPH solution was added; the mixture was incubated at 25 °C in the dark for 20 min, and the absorbance was measured at 517 nm. DPPH radical scavenging ability is expressed as mgTE / gDW.

[0047] 1.3.2.2 ABTS cationic radical scavenging ability Following existing methods with slight modifications, an ABTS cation radical scavenging experiment was conducted. An equal volume of 7.4 mmol / L LABTS solution was mixed with 2.6 mmol / L potassium persulfate solution and reacted in the dark for 14 h. The mixture was then diluted with 80% ethanol (v / v). 200 μL of the sample solution was added to 800 μL of the above working solution, and the mixture was incubated at 25°C in the dark for 6 min. The absorbance was measured at 734 nm. Results are expressed as mgTE / gDW.

[0048] 1.3.2.3 Ferrous Reduction Antioxidant Capacity (FRAP) The FRAP value was determined by referring to and optimizing existing methods. The FRAP working solution was prepared by three reagents in a volume ratio of 10:1:1: 178 mmol / L acetate buffer, 10 mmol / L 2,4,6-tripyridyltriazine (TPTZ) solution dissolved in 40 mmol / L hydrochloric acid, and 34 mmol / L ferric chloride solution. 1.8 mL of the FRAP working solution was mixed with 200 μL of the sample solution, and the mixture was reacted in the dark for 30 min. The absorbance was measured at 593 nm. The results are expressed as mgTE / gDW.

[0049] 1.4 Qualitative analysis of dietary fiber-bound phenols by UPLC-QTOF-MS The bound phenols obtained after treatment with 2M NaOH solution were qualitatively analyzed using UPLC-QTOF-MS. The chromatographic column was a Waters ACQUITY UPLC BEHC18 (2.1 × 50 mm, 1.7 μm). Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in acetonitrile solution. Flow rate: 0.3 mL / min, injection volume: 2 μL, column temperature: 35℃. Elution program: 0~0.5 min, 95% A; 0.5~8 min, 95-60% A; 8~15 min, 60-0% A; 15~17 min, 0% A; 17~17.1 min, 0-95% A; 17.1~20 min, 95% A. Mass spectrometry mass-to-charge ratio range: 100-3000 (m / z). MS scans were performed in negative ion mode.

[0050] 1.5 Characterization of the microstructure of dietary fiber The freeze-dried dietary fiber sample to be tested was adhered to the sample stage surface with conductive adhesive, and then vacuum sputtered with gold to improve conductivity. It was then placed in a scanning electron microscope and evacuated to a high vacuum. The accelerating voltage was set to 8-10 kV, and the focus and astigmatism were adjusted. The microstructure of the sample was observed in secondary electron mode, and images were taken and analyzed at different magnifications.

[0051] 1.6 In vitro simulation of gastrointestinal digestion and colonic fermentation 1.6.1 Simulating gastrointestinal digestion Dietary fiber with different binding degrees was mixed with pure water at a ratio of 1:30 (w / v), and the pH was adjusted to 2 with 3 mol / L HCl. 200 μL of 72 mg / mL pepsin solution was added and gently shaken. The mixture was incubated at 37 °C and 180 rpm for 2 h to complete gastric digestion. The pH was then adjusted to 6.5, and 4.5 mL of 10 mg / mL trypsin solution and 1.5 mL of 2 mg / mL bile salt solution were added sequentially and gently inverted to mix. The pH was then adjusted to 7.4 to simulate small intestinal digestion, and the mixture was incubated again at 37 °C and 180 rpm for 2 h to complete intestinal digestion. The solutions after each digestion stage were centrifuged (4 °C, 10000 rpm, 10 min), and the total phenol content and antioxidant activity of the supernatant were determined.

[0052] 1.6.2 Simulated Colon Fermentation Fresh stool samples were collected from three volunteers aged 23-24 years (2 males and 1 female), none of whom had a history of intestinal diseases. The fresh stool samples were mixed with PBS at a ratio of 1:5 (w / v), filtered through sterile gauze, and a fecal bacterial suspension was obtained. Preparation of BCM fermentation medium: L-cysteine ​​0.5g, cholic acid 0.5g, 0.025g / 100mL resazurin 4mL, 2mL Tween 80, yeast extract 2g, peptone 2g, NaCl 0.1g, K2HPO4 0.04g, KH2PO4 0.04g, MgSO4•7H2O 0.01g, CaCl2•2H2O 0.01g, NaHCO3 2g. Sterilize at 121℃ for 20min. Then add reducing agent: Na2S•9H2O 312.5mg, L-cysteine ​​312.5mg, mix thoroughly, and then add 10mL of 5mg / mL heme chloride solution and 0.5mL of 1% vitamin K1 solution respectively.

[0053] Four mL of the fermentation broth after gastrointestinal digestion (equivalent to 100 mg of dietary fiber with different polyphenol release levels) was added to 9.5 mL of BCM and 1.5 mL of fecal bacterial suspension to prepare an anaerobic fermentation broth. Samples were collected at 0.25 h, 6 h, 12 h, 24 h, and 48 h of anaerobic fermentation, with each time point repeated three times. After centrifugation, the supernatant and precipitate were collected and stored at -80℃. The supernatant was used to determine the total phenol content, antioxidant activity, and short-chain fatty acid content in the sample, while the precipitate was used to analyze the species and abundance of microorganisms in the feces.

[0054] 1.7 Determination of Short-Chain Fatty Acid Content Centrifuge the supernatant of the colonic fermentation broth from section 1.6.2 again (10000 rpm, 4℃, 5 min), take 400 μL of the supernatant from the second centrifugation, acidify at 4℃ for 1 h; add 400 μL of ethyl acetate, vortex for 5 min, let stand for 10 min, then centrifuge at 4℃ (10000 rpm, 4℃, 5 min); take the upper organic phase and store at -20℃ for later use.

[0055] The short-chain fatty acid content of colon fermentation samples after 48 h was determined by gas chromatography (Shimadzu GC-2030). The chromatographic column was an AT-FFAP (30m*0.32mm*0.5μm), with a split ratio of 10:1. The injection volume was 1μL, and the constant flow rate was 1mL / min. The temperature program was as follows: 105℃ for 3 min, increased to 170℃ at 5℃ / min, then increased to 230℃ at 15℃ / min and held for 3 min, for a total run time of 23 min. The injection port temperature and detector port temperature were both 240℃, and nitrogen was used as the carrier gas.

[0056] 1.8 High-throughput sequencing analysis of the effects of dietary fiber with different polyphenol release levels on fecal microbiota Genomic DNA was extracted from samples after 48 hours of colonic fermentation using the MagPure Soil DNA LQ Kit (Magan) according to the manufacturer's instructions. Purity was assessed by measuring the A260 / A280 ratio using a NanoDrop 2000 (Thermo Fisher Scientific, USA) spectrophotometer. DNA concentration and purity were detected by agarose gel electrophoresis, and the extracted DNA was stored at -20°C. Using the extracted genomic DNA as a template, PCR amplification of the bacterial 16S rRNA gene V3-V4 region was performed using barcode-specific primers and Takara Ex Taq high-fidelity enzyme. Universal primers 343F (5'-TACGGRAGGCAGCAG-3') and 798R (5'-AGGGTATCTAATCCT-3') were used. PCR products were detected by agarose gel electrophoresis. The purified DNA was then purified using AMPureXPbeads magnetic beads and used as a template for a second round of PCR amplification. The product was purified again using magnetic beads, and the purified second-round product was quantified using Qubit sequencing. The concentration was then adjusted for sequencing. Sequencing was performed using the Illumina NovaSeq 6000 sequencing platform, generating 250 bp paired-end reads. Sequencing was conducted by Shanghai Ouyi Biotechnology Co., Ltd. α- and β-diversity analyses and community structure distribution analyses were performed using QIIME2 software.

[0057] Example 2 2.1 Effects of alkaline hydrolysis on total phenolic content and antioxidant activity of KPDF See Figure 1 The release of dietary fiber and its bound phenols was regulated by controlling the concentration of NaOH. Results showed that increasing the NaOH concentration from 2M to 8M significantly improved the extraction rate of total phenols, increasing the total phenol content from 5.94±0.49 mgGAE / gDW to 15.57±0.19 mgGAE / gDW. However, when the NaOH concentration was further increased from 8M to 10M, the total phenol content of the extract only increased slightly, with no significant difference. This phenomenon may be because higher concentrations of NaOH solution can rapidly and efficiently disrupt the ester and ether bonds between the target extract and the cell wall matrix, thereby releasing more bound phenols; however, when the concentration reaches 8M, the disruptable bond structures have been largely utilized, so further increases in concentration no longer significantly improve the extraction rate.

[0058] The antioxidant activity of KPDF after alkaline hydrolysis was evaluated using three methods: DPPH, ABTS, and FRAP. The results are as follows: Figure 2As shown, when the NaOH concentration increased from 2M to 8M, the antioxidant activity of dietary fiber increased significantly. At the NaOH concentration of 8M, the antioxidant activity of DPPH was 38.92±1.43 mgTE / gDW, the antioxidant activity of ABTS was 47.12±2.43 mgTE / gDW, and the reducing power of FRAP was 23.83±0.32 mgTE / gDW. When the NaOH concentration continued to increase, its antioxidant activity did not change significantly.

[0059] The correlation analysis results between total phenols and antioxidant activity are shown in Table 1. The total phenol content of dietary fiber showed a highly significant positive correlation with DPPH, ABTS, and FRAP (p<0.01), with correlation coefficients (r) all above 0.990. This indicates that phenols are the main contributing components to the antioxidant activity of this dietary fiber, and their content changes are highly synchronized with the improvement of antioxidant capacity. Simultaneously, the three antioxidant evaluation indicators also showed a highly significant positive correlation (p<0.01), indicating a high degree of consistency in the antioxidant activity results measured by different methods.

[0060] Table 1: Correlation analysis between total phenol content and antioxidant activity after alkaline hydrolysis treatment of KPDF * indicates a significant correlation (p < 0.05); ** indicates a highly significant correlation (p < 0.01). 2.2 Effects of Alkaline Hydrolysis Combined with Ultrasonic Method on Total Phenolic Content and Antioxidant Activity of KPDF See Figure 3 During the ultrasonic treatment of dietary fiber samples, ultrasound waves can induce cell and cell wall expansion and rupture through cavitation and mechanical action. This further disrupts the bound structure of dietary fiber and intracellular polyphenols on the basis of alkaline hydrolysis, weakening the binding degree and allowing the intracellular active substances bound in the dietary fiber to fully dissolve and release, thus achieving efficient polyphenol extraction. Based on the changes in total phenols and their antioxidant activity after alkaline hydrolysis of KPDF, an optimal NaOH concentration of 8M was selected, with ultrasonic time controlled as a single variable, to further investigate the release of phenols bound to dietary fiber. Figure 3 It was observed that as the ultrasound treatment time increased from 15 min to 45 min, the total phenol content gradually increased, from 14.86 ± 0.72 mg GAE / g DW to 22.52 ± 0.45 mg GAE / g DW. However, after extending the ultrasound treatment time from 45 min to 75 min, there was no significant difference in the total phenol content. This may be because the destructive effect of ultrasound on the structure of dietary fiber has certain limits. After the release of polyphenols that are weakly or moderately bound to dietary fiber, some polyphenols remain tightly bound to dietary fiber by strong covalent bonds or hydrogen bonds. Further extending the ultrasound treatment time at this point makes it difficult to disrupt these high-binding-energy interactions, thus the content of bound polyphenols no longer increases significantly.

[0061] Changes in the antioxidant activity of KPDF treated by alkaline hydrolysis combined with ultrasonication are as follows: Figure 4 As shown, the antioxidant activity of dietary fiber increased with increasing sonication time from 0 min to 45 min. At 45 min, the antioxidant activity of DPPH was 46.41 ± 0.76 mg TE / g DW, the antioxidant activity of ABTS was 65.60 ± 4.31 mg TE / g DW, and the reducing power of FRAP was 29.45 ± 1.55 mg TE / g DW. Further increases in sonication time did not significantly alter the antioxidant activity. The trends of antioxidant activity measured by the three methods with increasing sonication time showed a high degree of consistency.

[0062] The correlation analysis results between total phenols and antioxidant activity are shown in Table 2. The total phenol content of the extract obtained by alkaline hydrolysis combined with ultrasonication of dietary fiber showed a highly significant positive correlation with DPPH, ABTS, and FRAP (p<0.01), indicating that phenolic substances are the main contributors to antioxidant activity, and their content changes are highly synchronous with the improvement of antioxidant capacity. Significant positive correlations were also observed among the three antioxidant evaluation indicators. The correlation coefficient between DPPH and ABTS was 0.913 (p<0.05), and the correlations among the other indicators were all highly significant (p<0.01), indicating good consistency among the antioxidant activity results measured by different methods. Based on the results of alkaline hydrolysis and alkaline hydrolysis combined with ultrasonication on the total phenols and antioxidant activity of KPDF, four dietary fibers with different polyphenol release levels—KPDF (untreated dietary fiber), A2M (2M NaOH solution without ultrasonication), A8M dietary fiber (8M NaOH solution without ultrasonication), and A8MUS dietary fiber (8M NaOH solution with ultrasonication for 45 min)—were selected for subsequent experimental determinations.

[0063] Table 2: Correlation analysis between total phenols and antioxidant activity after KPDF treatment by alkaline hydrolysis combined with ultrasonic method * indicates a significant correlation (p < 0.05); ** indicates a highly significant correlation (p < 0.01). 2.3 Qualitative analysis of bound polyphenols by UPLC-QTOF-MS Qualitative analysis of KPDF-bound phenols was performed using UPLC-QTOF-MS, as shown in Table 3. These included caffeic acid, 4-hydroxybenzoic acid, p-coumaric acid, ferulic acid, protocatechuic acid, salicylic acid, catechin, epicatechin, gallocatechin, epigallocatechin, dihydromyricetin, quercetin, dihydroquercetin, kaempferol, proanthocyanidins B1, and proanthocyanidins B2.

[0064] Furthermore, according to the research of Aamina Alim et al., the main phenolic substances in kiwifruit peel are catechins, epicatechins, epigallocatechins, and quercetin. This is consistent with the identification results of this study, indicating that the composition of phenolic compounds bound to dietary fiber in kiwifruit peel and pomace has a certain degree of universality, providing a solid theoretical basis and data support for subsequent research on the bioactivity of KPDF-bound phenols and the development and utilization of their resources.

[0065] Table 3. Determination of KPDF-bound phenols by UPLC-QTOF-MS in negative ion mode 2.4 Microstructure of dietary fiber with different polyphenol release levels The microstructures of KPDF, A2M, A8M, and A8MUS dietary fibers were observed using SEM, and the results are as follows: Figure 5-8 As shown. KPDF dietary fiber ( Figure 5 The surface of A2M dietary fiber is intact, smooth, and dense, without obvious pores or damage, exhibiting a relatively regular blocky structure. The surface structure of A2M dietary fiber is relatively stable and has good integrity. Figure 6 The integrity of the sample surface was damaged to some extent, the structure became rough, and wrinkles and irregular damage appeared on the surface, indicating that low-concentration alkaline hydrolysis had a certain corrosive effect on the surface structure of dietary fiber. A8M dietary fiber ( Figure 7 The overall structure of dietary fiber is significantly damaged, the surface becomes highly rough, and numerous pores and fragmented structures appear, exposing the cellulose skeleton. This indicates that high-concentration alkaline hydrolysis can significantly destroy components such as hemicellulose and lignin in dietary fiber, making the fiber structure loose and porous. A8MUS dietary fiber ( Figure 8 The structural damage was further intensified, with finer fiber fragments, richer pore structure, and complete exposure of the cellulose skeleton, resulting in a more fluffy and porous microstructure. This result indicates that ultrasonic treatment and high-concentration alkaline hydrolysis have a synergistic effect, which can further disrupt the dense structure of dietary fiber and significantly alter its surface morphology and pore characteristics.

[0066] 2.5 Effects of simulated gastrointestinal and colonic fermentation on the total phenol content of dietary fiber-bound phenols During the digestive stage in the stomach, such as Figure 9 As shown, the release of bound polyphenols in the KPDF group was at an extremely low level, with a total phenol content of only 1.01±0.01mgGAE / gDW; the total phenol content of other bound dietary fibers was also at a low level during the gastric digestion stage, indicating that the acidic environment of the stomach makes it difficult to break the bonds between dietary fiber and phenolic substances, and the bound active ingredients are difficult to release.

[0067] After entering the small intestine digestion stage, the release of bound polyphenols in all groups increased significantly. The release of bound polyphenols in A8M and A8MUS reached 11.45±0.23 mg GAE / gDW and 13.29±0.09 mg GAE / gDW, respectively, which were significantly higher than those in the KPDF and A2M groups. This indicates that the small intestinal environment can significantly promote the release of loose fiber-bound polyphenols, but its promoting effect on tightly bound dietary fiber is not obvious.

[0068] After entering the colonic fermentation stage, the total phenol release in each group continuously increased with fermentation time, reaching a peak at 24 hours. The peak total phenol release of A8MUS was 27.4 ± 0.46 mg GAE / g DW, significantly higher than the other groups; KPDF showed the lowest release, with no significant increase at any time point. This may be because colonic anaerobic microorganisms can secrete various extracellular enzymes to fully degrade the polysaccharide backbone and glycosidic bonds of loosely bound dietary fiber, thereby releasing large amounts of bound phenolic substances encapsulated in the fiber network; while highly bound and dense dietary fiber, due to its tightly cross-linked structure, is difficult to be effectively decomposed by microbial enzymes, significantly limiting the release of active ingredients.

[0069] 2.6 Effects of simulated gastrointestinal digestion and colonic fermentation on the antioxidant activity of dietary fiber-bound phenols This study evaluated the changes in the antioxidant activity of dietary fiber-bound phenols with different polyphenol release levels during in vitro simulated gastrointestinal and colonic fermentation processes using three methods: DPPH, ABTS, and FRAP. The results are as follows: Figure 10-12 As shown in the figure, compared with phenolic substances released during gastric and small intestinal digestion, the active substances released during colonic fermentation exhibit stronger free radical scavenging capabilities, which is highly consistent with the aforementioned release trend of total phenols. DPPH showed a significant upward trend in the early stage of colonic fermentation, while A8MUS reached a peak of 40.62±1.74 mgTE / gDW at 24 h of colonic fermentation, followed by a significant decrease at 48 h. The trend of ABTS was similar to that of DPPH, reaching a maximum value of 113.07±2.09 mgTE / gDW at 24 h of colonic fermentation, and then significantly decreasing with prolonged fermentation time. FRAP also showed a pattern of first increasing and then decreasing, reaching a peak of 38.18±0.87 mgTE / gDW at 24 h of fermentation, and then gradually decreasing.

[0070] The results showed that although the release of bound polyphenols mainly originated from the pre-disruption of dietary fiber structure by alkaline hydrolysis and combined alkaline hydrolysis and ultrasonic treatment, colonic fermentation further promoted the sustained release of bound phenols, which was beneficial to enhancing their antioxidant activity.

[0071] 2.7 Effects of dietary fiber with different polyphenol release levels on fecal microbiota 2.7.1 Effects of dietary fiber with different levels of polyphenol release on fecal microbiota α and β diversity The effects of dietary fiber with different polyphenol release levels on fecal microbiota α diversity in a simulated colonic fermentation 48h system, such as Figure 13 As shown in the figure. Compared to the KPDF group, the A2M and A8M groups showed a slight increase in Shannon and Simpson indices, with no significant difference among the three groups. Compared to the KPDF group, the A8MUS group showed a significant increase in both Shannon and Simpson indices. The results indicate that alkaline hydrolysis and alkaline hydrolysis combined with ultrasonic treatment can improve the diversity and richness of fecal microbiota. Fecal microbiota β-diversity was analyzed using PCoA. Figure 14 As shown, the analysis results based on various distance matrices can be used to observe differences between individuals or groups. Compared with the KPDF group, other groups are farther away, indicating better grouping. Furthermore, the samples in the A8M and A8MUS groups are close together, while they are significantly separated from other groups, indicating that the A8M and A8MUS groups have inter-group similarity and significant separation from the other two groups. In conclusion, dietary fiber combined with phenols through different treatments can both enhance the species diversity of in vitro fermented fecal microbiota and significantly alter the microbial community composition, achieving differentiated separation of the microbial community structure among different groups.

[0072] 2.7.2 Effects of dietary fiber with different polyphenol release levels on the relative abundance of fecal microbiota Community structure, also known as a biological community, specifically refers to the collective organisms within a particular ecological environment that interact directly or indirectly with each other. Different microbial communities possess unique nutrient utilization patterns and metabolic functions. The species composition, abundance ratio, and distribution characteristics of a community are key factors determining the stability and ecological function of a micro-ecosystem. By analyzing the relative abundance of species at the phylum and genus levels, the structural characteristics of microbial communities can be directly characterized, and the changing patterns of micro-ecological homeostasis can be effectively evaluated. The results of differences in the composition of each group of microbial communities are as follows: Figure 15-16 As shown in the figures, in the KPDF and A2M groups, the abundance of Proteobacteria-associated pathogens was higher at the phylum level, while the abundance of Bacteroidetes, which have intestinal probiotic functions, was relatively lower. Bacteroidetes, as a core beneficial gut microbiota, can efficiently degrade complex carbohydrates such as dietary fiber, maintain the balance of the gut microbiota structure, inhibit the excessive proliferation of harmful bacteria, and play an important role in stabilizing the gut microecological homeostasis. Both the A8M and A8MUS groups effectively increased the relative abundance of Bacteroidetes and decreased the abundance of harmful Proteobacteria, but had no significant effect on the abundance of Firmicutes.

[0073] At the horizontal level, such as Figure 17-19As shown, *Bacteroides*, *Shigella*, and *Providence* were the most prevalent bacterial genera in the sample, with significant differences in their physiological functions. *Bacteroides* is a typical beneficial bacterium, capable of breaking down large dietary fibers into short-chain fatty acids, repairing the intestinal epithelial barrier, and antagonizing pathogenic bacteria adhesion. *Shigella* and *Providence*, both belonging to the Proteobacteria phylum, are opportunistic pathogens; excessive proliferation can easily induce intestinal inflammation and disrupt the gut microbiota balance. Regarding the beneficial *Bacteroides* genus, compared to the KPDF control group, the abundance in the A2M group increased slightly, while the A8M and A8MUS groups significantly increased its relative abundance. For the opportunistic pathogen *Shigella*, the KPDF group had the highest relative abundance, while the A2M group showed the best inhibitory effect, with a significant decrease in abundance. The abundance in the A8M and A8MUS groups rebounded compared to the A2M group, but remained lower than that in the KPDF group. Providencia bronchiseptica showed an abnormally high abundance in the A2M group, while the A8M and A8MUS groups significantly inhibited its proliferation and drastically reduced its abundance (p < 0.05). This microbial community regulation is presumably closely related to the high content and strong antioxidant activity of polyphenols released by alkali treatment and the combined alkali treatment and ultrasonic method. Polyphenols can promote the proliferation of beneficial bacteria and limit the growth of harmful bacteria by scavenging the oxidative stress environment of the system and improving the microenvironment for bacterial survival.

[0074] 2.8 Effects of dietary fiber with different polyphenol release levels on short-chain fatty acid content The contents of short-chain fatty acids in each group after 48 hours of simulated colonic fermentation are shown in Table 4. The contents of short-chain fatty acids in most treatment groups were higher than those in the KPDF group. Acetic acid was the main metabolite in the fermentation system, accounting for the highest proportion of all short-chain fatty acids. The acetic acid production in the A2M and A8MUS groups was significantly higher than that in the KPDF group; although the acetic acid content in the A8M group was significantly higher than that in the KPDF group, it was lower than that in the A2M and A8MUS groups. Propionic acid plays an important role in regulating cholesterol metabolism and intestinal homeostasis; its content in the A2M group was significantly higher than that in the A8M and A8MUS groups. Butyric acid, as the main energy source and key anti-inflammatory metabolite of colonic cells, showed no significant difference among the groups, indicating that all samples could provide stable energy support for the colonic epithelium. In addition, isobutyric acid and isovaleric acid were also detected after fermentation. The isobutyric acid content in the A8MUS group was significantly higher than that in the KPDF and A2M groups, indicating that the fermentation process of branched-chain amino acids was more active under this treatment.

[0075] Colonic microbiota can interact with polyphenols: on the one hand, altering the degree of binding between dietary fiber and polyphenols can regulate fecal microbiota structure, promote the proliferation of glycolytic probiotics, and thus increase the production of short-chain fatty acids; on the other hand, the generated short-chain fatty acids can, in turn, enhance the absorption and bioavailability of dietary polyphenols and their fecal microbial metabolites, strengthening the health effects of polyphenols. The significant increase in acetic acid content provides sufficient precursors for the subsequent production of propionic and butyric acids, while also creating a favorable environment for the conversion and bioavailability of polyphenols. In summary, the A8MUS group releases higher levels of bound polyphenols, which, through targeted regulation of gut microbiota structure, enriches glycolytic beneficial bacteria such as Bacteroides. These dominant bacterial groups can fully decompose dietary fiber and promote the production of short-chain fatty acids.

[0076] Table 4. Short-chain fatty acid content of dietary fiber after colonic fermentation for 48 hours, with different levels of polyphenol release. Different lowercase letters in the table represent statistically significant differences between data points, p < 0.05. Results are expressed as mean ± standard deviation, n = 3.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described with reference to preferred embodiments, those skilled in the art should understand that various changes in form and detail can be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A method for preparing kiwi fruit skin residue dietary fiber with different degrees of bound polyphenol release, characterized by, Includes the following steps: Kiwifruit peel residue freeze-dried powder was subjected to high-temperature α-amylase, alkaline protease and saccharifying enzyme treatment to remove starch and protein, and then washed with ethanol, washed with distilled water and freeze-dried to obtain kiwifruit peel residue dietary fiber KPDF. KPDF was mixed with NaOH solutions of different concentrations at a ratio of 1:30 (w / v), and the mixture was sealed in a container and hydrolyzed by shaking at room temperature to obtain kiwifruit peel residue dietary fiber with different degrees of polyphenol release.

2. The production method according to claim 1, characterized by, The hydrolysis time was 4 hours, and the rotation speed was 180 r / min.

3. The preparation method according to claim 1, characterized in that, The concentration of the NaOH solution is 2-10M, and the content of dietary fiber-bound phenols released from kiwifruit peel residue is 5.94-15.57 mg GAE / g.

4. The production method according to claim 3, characterized by, After hydrolysis in NaOH solution at a concentration of 8M, the dietary fiber was subjected to ultrasonic treatment for 0-75 minutes, resulting in the release of phenolic compounds from the dietary fiber, with a content of 14.86-22.52 mg GAE / g.

5. A kiwifruit peel residue dietary fiber with different polyphenol release levels prepared by the method described in any one of claims 1-4.

6. The kiwifruit peel and pomace dietary fiber with different degrees of polyphenol release according to claim 5, characterized in that, The total phenolic content of the dietary fiber in the kiwifruit peel residue was 22.52±0.45 mgGAE / gDW, the antioxidant activity of DPPH was 46.41±0.76 mgTE / gDW, the antioxidant activity of ABTS was 65.60±4.31 mgTE / gDW, and the reducing power of FRAP was 29.45±1.55 mgTE / gDW. The dietary fiber from the kiwifruit peel residue has a loose and porous microstructure. The total phenol release from the dietary fiber bound in the kiwifruit peel residue reached 13.29±0.09 mgGAE / gDW and 27.4±0.46 mgGAE / gDW or higher 24 hours after entering the small intestine digestion stage and the colon fermentation stage, respectively.

7. The application of the kiwifruit peel and pomace dietary fiber with different polyphenol release levels as described in claim 5 in the preparation of products for regulating intestinal flora, characterized in that, The regulation of gut microbiota includes at least one of the following effects: Increase the α and β diversity of fecal microbiota; Reduce the relative abundance of Proteobacteria; Increase the relative abundance of Bacteroides; Reduce the relative abundance of opportunistic pathogens such as Shigella and / or Providencia.

8. The application of the kiwifruit peel and pomace dietary fiber with different polyphenol release levels as described in claim 5 in the preparation of products for promoting the formation of short-chain fatty acids, characterized in that, The short-chain fatty acids include acetic acid and / or isobutyric acid.

9. The application according to claim 7 or 8, characterized in that, The product is a functional food ingredient or an intestinal microecological regulator.

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