Red date modified polysaccharide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of food processing, and particularly relates to red date modified polysaccharide as well as a preparation method and application thereof. Every 100 mg of the red date modified polysaccharide contains 34.77 mg + / -0.504 mg of total sugar, 32.00 mg + / -2.01 mg of uronic acid and 8.89 mg + / -0.22 mg of reducing sugar. The red date polysaccharide is modified through a low-temperature ultrasonic-assisted deep-eutectic solvent extraction technology and a directional enzyme digestion technology, and compared with the in-vitro intestinal flora adjusting capacity of the red date polysaccharide before and after modification, the intestinal flora of the red date modified polysaccharide obtained through modification has higher prebiotic activity.

Description

Technical Field

[0001] This application belongs to the field of food processing technology, specifically relating to a modified polysaccharide of jujube, its preparation method, and its application. Background Technology

[0002] Jujube (Ziziphus jujuba Mill.) is a ripe fruit belonging to the genus Ziziphus and the family Rhamnaceae. Since ancient times, jujubes have been listed as one of the "Five Fruits" (chestnut, peach, plum, apricot, and jujube), and are considered the "King of Tonics" among fruits, possessing extremely rich nutritional value. Jujubes are a food and medicine homology. Past research has shown that jujubes contain important bioactive substances with antioxidant, anti-fatigue, immune and intestinal metabolic regulation, anti-inflammatory, and liver-protective activities, such as polysaccharides, polyphenols, amino acids, nucleotides, fatty acids, dietary fiber, alkaloids, and other nutrients, with polysaccharides accounting for over 50%. Jujube polysaccharides, as a natural plant polysaccharide, also possess immunomodulatory, antitumor, antiviral, anti-inflammatory, antioxidant, and hypoglycemic activities, as well as effects on the intestinal mucosal immune system.

[0003] Research reports indicate that current research on jujube polysaccharides is still in the stage of focusing on extraction processes and the bioactivity of crude extracts. Studies on the relationship between the structural characterization and bioactivity of modified jujube polysaccharides are relatively limited. Many natural polysaccharides exhibit low or no bioactivity due to their high molecular weight and poor water solubility, restricting their application in many fields. Jujube polysaccharides, as a type of natural polysaccharide, also suffer from this problem. Modifying polysaccharides can reduce their molecular weight and improve their bioactivity. Currently, commonly used methods for polysaccharide molecular modification include chemical modification, physical modification, and biological modification. However, chemical modification methods often involve highly toxic catalysts, numerous byproducts, and difficulties in separation, resulting in significant side effects. Therefore, a method to address this issue is urgently needed. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a modified polysaccharide of jujube, its preparation method, and its application. Specifically, the following technical solution is adopted: In a first aspect, the present invention provides a modified polysaccharide of jujube, characterized in that the modified polysaccharide of jujube contains, per 100 mg, total sugar: 34.77 mg ± 0.504 mg, uronic acid: 32.00 mg ± 2.01 mg and reducing sugar: 8.89 mg ± 0.22 mg.

[0005] As a further preferred embodiment, the monosaccharides contained in the modified jujube polysaccharide include, by mole percentage, the following: Fructose 0.37%, rhamnose 4.69%, arabinose 10.83%, galactose 11.90%, gluconic acid 2.12%, xylose 1.24%, mannose 0.81%, galacturonic acid 66.97%, glucuronic acid 1.07%.

[0006] As a further preferred embodiment, the molecular weight of the jujube-modified polysaccharide is (0.322±0.001)×10⁻⁶. 4 Da - (215.835 ± 0.363) × 10 4 Da.

[0007] Secondly, the present invention provides a method for preparing the above-mentioned modified polysaccharide of jujube, comprising the following steps: Jujube polysaccharide was placed in α-galactosidase solution, the pH was adjusted to 5.5, and the reaction was carried out at 37℃ for 1 h to inactivate it. After centrifugation, the supernatant was collected, precipitated with alcohol, reconstituted, and freeze-dried to obtain the jujube-modified polysaccharide.

[0008] The α-galactosidase used in this invention is a glycoside hydrolase that selectively hydrolyzes the terminal α-1,6-glycosidic bonds in galactosidic oligosaccharides, polysaccharides, glycoproteins, and glycolipids. It can also catalyze the hydrolysis of the terminal α-galactose moiety in oligosaccharides and polysaccharides, aiding in the digestion of galactose, a common component in plant-based foods. It is industrially produced for use in food and feed processing, as well as in dietary supplements. This invention is the first to employ α-galactosidase-catalyzed modification of jujube polysaccharides as a biomodification method, exhibiting excellent specificity and selectivity, and minimizing the formation of undesirable byproducts.

[0009] As a further preferred embodiment, the jujube polysaccharide is prepared by the following process: S1. Dissolve the jujube powder in a eutectic solvent, then extract it using ultrasound. After extraction, centrifuge to obtain the supernatant. S2. The supernatant is added to ethanol for alcohol precipitation. After the alcohol precipitation is completed, the mixture is allowed to stand at 4°C. After standing, the precipitate is obtained by centrifugation. S3. The precipitate is reconstituted with water, then the protein is removed using Sevage reagent, dialyzed, and freeze-dried to obtain the jujube polysaccharide.

[0010] As a further preferred embodiment, the ratio of jujube powder to eutectic solvent is 1:25.

[0011] As a further preferred embodiment, the eutectic solvent is obtained by dissolving choline chloride and anhydrous citric acid in water, wherein the molar ratio of choline chloride to anhydrous citric acid is 1:1.

[0012] As a further preferred embodiment, the Sevage reagent is a mixed solution of chloroform and n-butanol in a volume ratio of 4:1.

[0013] As a further preferred embodiment, the extraction power during ultrasonic extraction is 130W-160W, the extraction temperature is 60℃-80℃, and the extraction time is 20 min-40 min.

[0014] Thirdly, the application of modified jujube polysaccharides in the preparation of prebiotic foods that regulate intestinal flora.

[0015] The beneficial effects of this invention are as follows: This invention modifies jujube polysaccharides using low-temperature ultrasound-assisted eutectic solvent extraction and directional enzymatic digestion techniques. The physicochemical properties, structural characteristics, and in vitro gut microbiota regulation capabilities of jujube polysaccharides before and after modification are compared. The modified jujube polysaccharides obtained by modification exhibit more prebiotic activity in the gut microbiota. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The image shows the high-performance gel permeation chromatogram of jujube polysaccharide and its modified products (Note: ZJP and α-ZJP represent jujube polysaccharide and modified jujube polysaccharide, respectively). Figure 2 The Fourier transform infrared spectra of jujube polysaccharide and its modified products are shown (Note: ZJP and α-ZJP represent jujube polysaccharide and modified jujube polysaccharide, respectively). Figure 3 The image shown is a scanning electron microscope image of jujube polysaccharide ZJP at 500x. Figure 4 The image shown is a scanning electron microscope image of jujube polysaccharide ZJP at 1000x. Figure 5 The image shown is a scanning electron microscope image of jujube polysaccharide ZJP at 2000x. Figure 6 The image shown is a scanning electron microscope image of the modified jujube polysaccharide product α-ZJP at 500x. Figure 7 The image shown is a scanning electron microscope image of the modified jujube polysaccharide product α-ZJP at 1000x. Figure 8 The image shown is a scanning electron microscope image of the modified jujube polysaccharide product α-ZJP at 2000x. Figure 9 The figure shows the pH changes during the in vitro fermentation of jujube polysaccharide and its modified products. Figure 10 The figure shows the changes in OD600 values ​​during the in vitro fermentation of jujube polysaccharide and its modified products. Figure 11 The figure shows the changes in uronic acid content during the in vitro fermentation of jujube polysaccharide and its modified products. Figure 12 The figure shows the changes in reducing sugar content during the in vitro fermentation of jujube polysaccharide and its modified products. Figure 13 The comparison shows the α-diversity indices (Chao1index, Shannon index, Simpson index, and Pielou-e index) of the gut microbiota after in vitro fermentation of jujube polysaccharide and its modified products; (where ZJP, α-ZJP, PC, and CK represent jujube polysaccharide, modified jujube polysaccharide, positive control, and blank control, respectively). Figure 14 The comparison of the β-diversity index (PCoAclustering analysis) of in vitro fermentation gut microbiota of jujube polysaccharide and its modified products is shown; (where ZJP, α-ZJP, PC, and CK represent jujube polysaccharide, modified jujube polysaccharide, positive control, and blank control, respectively). Figure 15 The figure shows the in vitro fermentation phylum-level gut microbiota species composition analysis of jujube polysaccharide and its modified products. Figure 16 The figure shows the relative abundance of gut microbiota at the in vitro fermentation level of jujube polysaccharide and its modified products. Figure 17 The image shows a heatmap analysis of the relative abundance of jujube polysaccharides and their modified products at the in vitro fermentation level compared to the gut microbiota; (Note: ZJP, α-ZJP, PC, and CK represent jujube polysaccharides, modified jujube polysaccharides, positive control, and blank control, respectively). Figure 18 The figure shows the species differences and biomarkers of intestinal flora in vitro after fermentation of jujube polysaccharide and its modified products (ASV / OTU Venn plot). Figure 19 The image shows the species differences and biomarkers of intestinal flora in the in vitro fermentation of jujube polysaccharide and its modified products (cladograms of different groups and taxa). Figure 20 The figure shown is a graph of species differences and biomarkers of intestinal flora in vitro after fermentation of jujube polysaccharide and its modified products (LDA score); (where ZJP, α-ZJP, PC, and CK represent jujube polysaccharide, modified jujube polysaccharide, positive control, and blank control, respectively). Detailed Implementation The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0018] Example 1 A modified polysaccharide for jujubes is prepared by the following steps: (1) Preparation of Jujube Polysaccharide S1. Preparation of jujube powder: Remove the pits from the jujubes, cut them into small pieces, dry them at 60℃ for 24-48 hours, grind them into powder, and pass them through a 60-mesh sieve. S2. Ultrasonic-assisted eutectic solvent extraction: The material-to-liquid ratio was 1:25 (w / v), the extraction power was 150W, the extraction temperature was 70℃, and the extraction time was 30min. The eutectic solvent consisted of choline chloride, anhydrous citric acid, and ultrapure water. The ratio of choline chloride to anhydrous citric acid was 1:1 according to the molar ratio. The water content of the eutectic solvent was 30%. After extraction, the mixture was centrifuged at 5000 rpm for 10min and the supernatant was retained. S3, alcohol precipitation: Add anhydrous ethanol to the extract, and after alcohol precipitation, let it stand overnight at 4°C; S4. Resolution: Remove the supernatant, centrifuge and retain the precipitate, then resolution with ultrapure water; S5. Deproteinization: Remove protein 5-8 times using the Sevage method, which uses a 4:1 (V:V) mixture of chloroform and n-butanol. S6. Dialysis: Use a dialysis bag to remove impurities. The molecular weight cutoff of the dialysis bag is 8000-14000 Da. Dialyze with tap water for two days, then continue dialysis with ultrapure water for one day. Finally, freeze dry to obtain jujube polysaccharide (ZJP).

[0019] (2) Preparation of modified polysaccharides from jujube α-galactosidase was prepared into a 1000 U / mL enzymatic hydrolysate. Jujube polysaccharide obtained in step (1) was prepared into a 10 mg / mL jujube polysaccharide solution. The jujube polysaccharide was added to the enzymatic hydrolysate at an activity of 2000 U / g, mixed well, and the pH was adjusted to 5.5 with 1M hydrochloric acid and 1M sodium hydroxide. The reaction was carried out at 37℃ for 1 h, inactivated at 100℃ for 10 min, and centrifuged at 12000 rpm for 10 min. The supernatant was collected. The supernatant was precipitated with alcohol and redissolved, and then freeze-dried to obtain jujube modified polysaccharide (α-ZJP) with reduced molecular weight.

[0020] Comparative Example 1 To investigate the effects of different enzymes on the efficacy, the modification effects of five enzymes, including α-galactosidase, on jujube polysaccharides were specifically studied, as follows: Xylanase 100,000 U / g, β-mannan 50,000 U / g, cellulose 10,000 U / g, and rhamnose 30,000 U / g were used to prepare a 10 mg / mL solution of jujube polysaccharides. Each enzyme was added to the enzyme hydrolysate at an activity of 400 U / g, mixed, and reacted for 1 h under the optimal conditions for each enzyme (Table 1). The enzymes were then inactivated at 100℃ for 10 min, centrifuged at 12,000 rpm for 10 min, and the supernatant was collected. The supernatant was precipitated with alcohol, redissolved, and then freeze-dried to obtain the corresponding modified polysaccharides. The molecular weight changes of jujube polysaccharides modified by different enzymes were compared using high-performance liquid chromatography (Table 2). Table 2 shows that only under the action of α-galactosidase did the molecular weight change significantly, mainly in Mw2, which decreased from over 800 kDa to 270 kDa.

[0021] Table 1 Optimal reaction conditions for various enzymes Table 2. Changes in molecular weight of jujube polysaccharides under the action of different enzymes. Note: Mw1, Mw2, Mw3, Mw4, and Mw5 are the five molecular weight components of jujube polysaccharide.

[0022] Comparative Example 2 To investigate the effect of α-galactosidase at different enzyme activities on the molecular weight of jujube polysaccharides, enzyme addition levels of 400 U / g, 800 U / g, 1200 U / g, and 2000 U / g were set. The pH was adjusted to 5.5 using 1M hydrochloric acid and 1M sodium hydroxide, the reaction was carried out at 37℃ for 1 h, followed by inactivation at 100℃ for 10 min, and centrifugation at 12000 rpm for 10 min. The supernatant was collected, precipitated with alcohol, redissolved, and then freeze-dried to obtain the jujube polysaccharides under the corresponding enzyme activity conditions, as shown in Table 3. It can be seen that with the increase of α-galactosidase addition, Mw3 also showed some changes, but the molecular weight of Mw2 still decreased. Table 3. Changes in molecular weight of jujube polysaccharides under different amounts of α-galactosidase addition. Example 2 The physicochemical properties of the jujube-modified polysaccharide prepared in Example 1 were characterized as follows: (1) Determination of the chemical composition of modified polysaccharides from jujube Total sugar content was determined by phenol-sulfuric acid method, using glucose as the standard. Protein content was determined by Coomassie brilliant blue method using bovine serum albumin as the standard. Glucuronic acid content was determined by carbazole-sulfuric acid method using galacturonic acid as the standard.

[0023] Reducing sugars were determined by colorimetry. For details, please refer to: Authors: Hu Yunfei, Zhou De, Zeng Qinglan, Jin Song, Zhao Ming, Tang Qingyan; Title: Optimization of Enzymatic Hydrolysis Process, Structural Characterization and Antioxidant Activity Analysis of Gastrodia elata Polysaccharide; Journal: Science and Technology of Food Industry, Volume 46, Issue 04, Pages 84-90; DOI: 10.13386 / j.issn1002-0306.2024030247.

[0024] The chemical compositions of jujube polysaccharide (ZJP) and modified jujube polysaccharide (α-ZJP) are shown in Table 4. The total sugar content of ZJP is significantly higher than that of α-ZJP, indicating that α-ZJP causes the destruction or degradation of some polysaccharide structures, resulting in a decrease in total sugar content. The protein content of α-ZJP is reduced by nearly half (47%). This may be because α-galactosidase treatment of ZJP removes some bound proteins, or the proteins undergo hydrolysis or denaturation during treatment, leading to a decrease in content. The reducing sugar content of α-ZJP is significantly increased: it is 2.61 times that of ZJP, an increase of more than 1.6 times. Its increased content may be due to the α-galactosidase treatment causing the polysaccharide chains in the total sugar to break down, generating more small-molecule reducing sugars, consistent with the decreasing trend of total sugar, i.e., large-molecule sugars are decomposed into small-molecule reducing sugars. The uronic acid content of α-ZJP is significantly higher than that of ZJP, increasing by about 47%. Its increased content may be due to the increased exposure of uronic acid in the polysaccharide after α-galactosidase treatment (α-ZJP). The results showed that the structure of jujube polysaccharide was modified by low-temperature ultrasound-assisted eutectic solvent extraction and directional enzymatic digestion, and α-galactosidase had a significant regulatory effect on the chemical composition of ZJP.

[0025] Table 4 Chemical composition of jujube polysaccharides and their modified products Note: ZJP and α-ZJP represent jujube polysaccharide and modified jujube polysaccharide, respectively. The results in the table are expressed as mean ± standard deviation (Mean ± SD) when n=3.

[0026] (2) Structural characteristics of modified polysaccharides from jujube To further determine the primary structural characteristics of jujube polysaccharides and modified jujube polysaccharides (ZJP, α-ZJP), their molecular weight, monosaccharide composition, functional groups, and morphology were studied using high performance liquid chromatography, ion chromatography, Fourier transform infrared spectroscopy, nanoparticle size potentiometer, and cold field emission scanning electron microscopy, thereby determining whether their modification was successful.

[0027] The molecular weight of modified polysaccharide from jujube was determined using high-performance gel permeation chromatography (Agilent 1260 Infinity II LC System). Specific methods are described in the following table: Authors: Changping Zheng, Qun Dong, Huanjun Chen, Qifei Cong, Kan Ding; Title: Structural characterization of a polysaccharide from Chrysanthemum morifolium flowers and its antioxidant activity; Journal: Carbohydrate Polymers, Volume 130, Pages 113-121; DOI: 10.1016 / j.carbpol.2015.05.004.

[0028] The result is as follows Figure 1 As shown, Figure 1 High-performance gel permeation chromatograms of ZJP and α-ZJP are shown. The results showed that ZJP and α-ZJP are heteropolysaccharides containing five molecular weights: ZJP (2146.131±3.539 kDa, 884.480±2.461 kDa, 51.313±0.407 kDa, 17.690±0.204 kDa, 3.375±0.012 kDa) and α-ZJP (2158.353±3.628 kDa, 127.924±0.645 kDa, 35.255±0.040 kDa, 18.460±0.010 kDa, 3.216±0.007 kDa). The gel permeation chromatograms of ZJP and α-ZJP showed shifts, with peak responses increasing as molecular weight decreased. The peak around 884 kDa was not detected in α-ZJP, while the detected molecular weight for α-ZJP was 127 kDa. The peak around kDa indicates that the molecular weight of jujube polysaccharide changed after being modified by directional enzymatic digestion.

[0029] Note: ZJP and α-ZJP represent jujube polysaccharide and modified jujube polysaccharide, respectively. The results in the table are expressed as mean ± standard deviation (Mean ± SD) when n=3.

[0030] (3) Monosaccharide composition analysis To reveal the effect of enzymatic modification on the chemical structure of jujube polysaccharides, the monosaccharide composition, chemical groups, and morphology of jujube polysaccharides were systematically studied. The constituent sugars of ZJP and α-ZJP were obtained by HPAEC-PAD analysis. The results showed that the monosaccharide compositions of ZJP and α-ZJP were consistent, consisting of galacturonic acid (Gala), galactose (Gal), arabinose (Ara), rhamnose (Rha), xylose (Xyl), fructose (Fuc), glucose (Glc), glucuronic acid (Glca), and mannose (Man). The results indicated that after modification with targeted enzymatic digestion, the types of constituent sugars in jujube polysaccharides remained unchanged, but the proportions of these sugars altered, with an increase in the proportion of uronic acid leading to a slight decrease in the proportion of neutral sugars.

[0031] The monosaccharide composition of the modified polysaccharide from jujube was determined using ion chromatography (ICS5000). Specific methods are detailed in the following table: Authors: Haibin Zhu, Liuming Xie, Weidong Zhang, Qiang Yu, Yi Chen, Jianhua Xie; Title: Extraction and properties of Cyclocarya paliurus leaf polysaccharide and its phosphorylated derivatives; Journal: Industrial Crops and Products, Volume 210; DOI: 10.1016 / j.indcrop.2024.118205. The results are shown in Table 2. Table 5. Monosaccharide composition of jujube polysaccharides and their modified products. (4) Infrared spectral characteristics The functional groups of the modified jujube polysaccharide were analyzed using Fourier transform infrared spectroscopy (Nicolet 5700). The structural changes of the jujube polysaccharide before and after modification were further investigated using FT-IR spectroscopy.

[0032] The result is as follows Figure 2 As shown, the infrared spectra of ZJP and α-ZJP are similar, both exhibiting typical absorption bands typical of pectin-based acidic polysaccharides. Their FT-IR spectra are located at 3427.9 cm⁻¹ and 3430.3 cm⁻¹, respectively. -1 This is due to the stretching vibration of -OH, and 2936.2 cm. -1 and 2928.9 cm -1 The corresponding absorption peak at 1606.7 cm⁻¹ is the peak value of the CH tensile vibration. -1 and 1736.2 cm -1This is mainly due to the presence of C=O and COOH. 1000 cm -1 Up to 1200 cm -1 The absorption peak at 822.9 cm⁻¹ is due to the stretching vibrations of the CO and C C bonds in the pyranose ring. The peak at 822.9 cm⁻¹ is caused by the presence of α-glycosidic bonds. Each of the two polysaccharides shows an absorption peak at 600 cm⁻¹. -1 Up to 650 cm -1 Each peak shows a value, indicating tensile vibrations associated with the pyranose rings in the glycan backbone.

[0033] (5) Particle size and potential A 1 mg / mL solution of jujube polysaccharide was prepared, and the particle size and potential were measured using a Malvern PAalytical / Zetasizer Pro nanoparticle size potentiometer. The results are shown in Table 6.

[0034] Table 6 Particle size and potential The results in the table above show that the particle size of α-ZJP is significantly reduced, which can increase its ability to contact the substrate and improve its utilization rate. Both ZJP and α-ZJP are very stable, indicating that they can be well applied in actual production.

[0035] (6) Scanning electron microscope To further reveal the morphological characteristics of the modified jujube polysaccharide, scanning electron microscopy was performed.

[0036] The result is as follows Figures 3-8 This indicates that after being treated with α-galactosidase, ZJP changed from a smooth, silky sheet-like structure to a porous, tightly packed, sponge-like shape. This demonstrates that the morphology of ZJP changed significantly after modification with α-galactosidase.

[0037] Example 3 This study investigated the in vitro fermentation characteristics of jujube polysaccharides and their modified products, as well as their regulatory effects on gut microbiota. I. Experimental Methods: 1. Materials and reagents (as shown in Table 7) Table 7 Materials and reagents for in vitro fermentation assay 2. In vitro simulated gut microbiota fermentation experiment: a. Intestinal Microbiota Collection: Fresh fecal samples were collected from seven healthy volunteers (aged 22-26, four males and three females). All volunteers had no history of gastrointestinal diseases, maintained good dietary habits, and had not taken antibiotics or other medications, or consumed foods or medications containing probiotics or prebiotics within the past three months. Samples were collected simultaneously from all seven volunteers at a pre-agreed timeframe. The sample size was determined based on the number of fermentation samples. Immediately after collection, the fecal samples were transferred to an anaerobic glove box and diluted 1:4 (w / v) with PBS buffer containing 0.1% L-cysteine ​​hydrochloride. The samples were vortexed, allowed to stand for 5 minutes, and the supernatant was passed through four layers of sterile gauze to obtain fecal filtrate. Equal volumes of fecal filtrates from six volunteers were then mixed.

[0038] b. Culture medium preparation: Each 1 L of culture medium contains: 2 g peptone, 2 g yeast extract powder, 0.1 g NaCl, 0.04 g KH2PO4, 0.04 g K2HPO4, 0.01 g MgSO4·7H2O, 0.01 g CaCl2·6H2O, 2 g NaHCO3, 0.5 g L-cysteine ​​hydrochloride, 0.5 g bile salts, 10 μL vitamin K1, 2 mL Tween 80, and 0.005 g heme chloride. After dissolving and mixing, adjust the pH to 7.3. Then sterilize by nitrogen blowing and at 121 ℃ for 15 min.

[0039] c. Mixed Culture: In an anaerobic environment (5% H2, 5% CO2, 90% N2), add 2% (v / v) mixed fecal filtrate to a Hungate anaerobic tube using a 1 mL syringe (the interval between fecal collection and culture should not exceed 2 h). Incubate on a shaker at 37 ℃. At six time points (0, 6, 12, 24, 36, and 48 h), remove the anaerobic tubes and quickly transfer them to sterile EP tubes. Immediately freeze at -80 ℃. Centrifuge 1 mL of fermentation broth at 12000 rpm for 5 min. Use the supernatant to measure pH and the precipitate to extract DNA. Inulin and carbon-free medium serve as positive and negative controls, respectively. A separate anaerobic tube is used for each time point, and each time point includes three independent parallel experiments.

[0040] 3. Determination of pH and OD600 of fermentation broth: The turbidity of each culture medium was measured at 600 nm using an ELISA reader at six time points: 0, 6, 12, 24, 36, and 48 h. The pH was determined using a pH meter.

[0041] 4. Determination of uronic acid: Take 0.1 mL of the supernatant of the fermentation broth and dilute it 50 times. Use glucuronic acid as a standard curve and determine the concentration using the carbazole-sulfuric acid method. For specific methods, please refer to: Authors: Weiwei Li, Jingya Wang, Zhongqin Chen, Xudong Gao, Yue Chen, Zihan Xue, Qingwen Guo, Qiqi Ma, Haixia Chen; Title: Physicochemical properties of polysaccharides from Lentinus edodes under highpressure cooking treatment and its enhanced anticancer effects; Journal: International Journal of Biological Macromolecules, Volume 115; Pages 994-1001; DOI:10.1016 / j.ijbiomac.2018.04.094. 5. Neutral sugar determination: Take 0.1 mL of the supernatant of the fermentation broth and dilute it 50 times. Use glucose as a standard and determine the total carbohydrate amount using the phenol-sulfuric acid method. For details, please refer to: Author: Mauro Mecozzi; Title: Estimation of total carbohydrate amount in environmental samples by the phenol–sulphuric acid method assisted by multivariate calibration; Journal: Chemometrics and Intelligent Laboratory Systems, Volume 79, Issue 1-2, Pages 84-90; DOI: 10.1016 / j.chemolab.2005.04.005. 6. Determination of SCFAs content: 1 µL of the sample solution was injected into a gas chromatograph (7890B) equipped with a capillary column using an autosampler for detection. The capillary column model and specifications were: Agilent 19091F-433, HP-FFAP, 30m × 250 µm × 0.25 µm. The determination conditions and program were as follows: injection port temperature 220℃; detector temperature 250℃; FID flame ionization detector; temperature program: initial column oven temperature 60℃, hold for 5 min, increase to 160℃ at 10℃ / min, hold for 2 min, increase to 220℃ at 20℃ / min, hold for 5 min; carrier gas was nitrogen, flow rate 30 mL / min, split ratio 10:1; fuel gas was hydrogen, flow rate 35 mL / min; air flow rate was 400 mL / min. For specific methodology, please refer to: Authors: Guohua Zhao, Margareta Nyman, Jan Åke Jönsson; Title: Rapid determination of short-chain fatty acids incolonic contents and faeces of humans and rats by acidified water-extraction and direct-injection gas chromatography; Journal: Biomedical Chromatography, Volume 20, Issue 8, Pages 674-682; DOI: 10.1002 / bmc.580. Note: CK, PC, ZJP, and α-ZJP groups represent the blank control (no carbon source), positive control (inulin), jujube polysaccharide, and jujube modified polysaccharide groups, respectively. Results in the table are expressed as mean ± standard deviation (Mean ± SD) when n=3. Uppercase letters indicate significant differences (p<0.05) between different time points within the same group, while lowercase letters indicate significant differences (p<0.05) between different groups at the same time point. ND = Not detected.

[0042] 7.16S rRNA gene sequencing: After 48 h of fermentation, the fermentation broths of each group were centrifuged, and the precipitates were stored at -80°C for the determination of microbial community structure. The V3-V4 region of bacterial 16S rRNA in each sample was amplified and analyzed. After obtaining OTUs, changes in the microbial community structure of the CK, PC, ZJP, and α-ZJP groups were measured, and α-diversity and β-diversity of the intestinal flora were analyzed.

[0043] Note: CK, PC, ZJP, and α-ZJP groups represent the blank control, positive control, jujube polysaccharide, and modified jujube polysaccharide groups, respectively. Results in the figure are represented as n=3. p-value <0.05 indicates significant difference.

[0044] II. Experimental Results: 1. pH and OD600 of the fermentation broth Figure 9 The pH changes of the fermentation broth over 48 hours were shown. The initial pH of the fermentation in all groups was >7.2, and it decreased to varying degrees during fermentation. At each time point (6, 12, 24, and 48 h), the pH values ​​of the polysaccharide and PC groups were significantly lower than those of the control group (p<0.01). Furthermore, at 6 h of fermentation, the pH value of the PC group rapidly decreased from 7.58±0.011 to 5.73±0.03, a decrease of 24.4%, while the pH values ​​of the ZJP and α-ZJP groups were only 12.1% and 14.5%, respectively. At 12 h of fermentation, the pH of the polysaccharide group reached its lowest point, while the pH values ​​of the ZJP and α-ZJP groups were 5.96±0.01 and 6.24±0.01, respectively. After 48 hours of fermentation, the pH values ​​of the PC (4.46±0.05) and ZJP, α-ZJP (5.96±0.01, 6.24±0.01) groups were significantly lower than those of the CK group (7.15±0.01). Throughout the fermentation process, the pH value of the PC group decreased significantly and was lower than that of the other three groups at every time point; the pH value of the polysaccharide group decreased significantly from 0 to 12 and increased significantly from 12 to 48. The significant decrease in pH value of the PC and polysaccharide groups at different fermentation stages may be due to the high molecular weight of ZJP and α-ZJP; the slower decrease in pH value of the CK group may be due to the lack of available carbohydrates in the culture medium. In conclusion, these data indicate that the decrease in pH value of the fermentation broth is mainly due to the accumulation of acidic substances such as SCFA, and the pH of ZJP and α-ZJP maintained at 6.0-6.5 may be more suitable for most probiotics (such as butyric acid-producing bacteria).

[0045] Figure 10This study illustrates the changes in the total number of microorganisms in different groups of the in vitro fermentation mixture over 48 hours. At 6 h of fermentation, the OD600 value of the CK group was significantly lower than that of the PC and polysaccharide groups. The rate of increase in OD600 value occurred moderately within 48 hours after fermentation, indicating that microbial growth rate was limited in the absence of an effective carbon source. Furthermore, the PC group showed the fastest microbial growth rate at 12 h of fermentation, with the OD600 value increasing from 0.77 ± 0.02 to 1.16 ± 0.03. However, after 12 hours, the microbial growth rate slowed down, possibly due to pH inhibition. The ZJP group showed the fastest microbial growth rate at 12 h, increasing from 0.72 ± 0.01 to 1.00 ± 0.01, significantly lower than the inulin group (p < 0.05). It continued to rise slightly after 48 h, possibly related to the milder pH environment (6.11). The α-ZJP group showed the fastest microbial growth rate at 6 h, increasing from 0.46 ± 0.04 to 0.90 ± 0.03, significantly higher than the inulin group (p < 0.05), corresponding to the fastest consumption of neutral sugars. However, the growth rate stagnated from 12 to 24 h and then rose again after 48 h, possibly indicating staged substrate utilization. Furthermore, after 48 hours, the OD600 values ​​of the ZJP and α-ZJP groups (1.13±0.02, 1.18±0.02) were not significantly different from those of the inulin group (1.14±0.02), while the OD600 value of the CK group (0.66±0.02) was significantly lower than that of the other three groups (p<0.0001). This indicates that inulin and polysaccharide groups promoted the growth rate of gut microbiota and were well utilized by the microorganisms. The α-ZJP group showed rapid early proliferation and efficient sugar utilization, but its SCFA production was slightly lower than that of ZJP, possibly due to different metabolic pathways.

[0046] 2. Changes in neutral sugars and uronic acids Figures 11-12The results show that the neutral sugar content of different fermentation mixtures changed during 48 h of fermentation. At 0 h of fermentation, the neutral sugar content of the CK (0.28 ± 0.02 mg / mL), PC (4.91 ± 0.21 mg / mL), ZJP (2.34 ± 0.05 mg / mL), and α-ZJP (1.83 ± 0.09 mg / mL) groups differed significantly (p<0.0001). After 6 h of fermentation, the neutral sugar content of the CK, PC, ZJP, and α-ZJP groups decreased by 71.4%, 18.1%, 46.2%, and 59.6%, respectively. After 12 h of fermentation, the decrease in neutral sugar content of the CK, PC, ZJP, and α-ZJP groups was 78.6%, 35.6%, 77.8%, and 83.6%, respectively. After 24 h of fermentation, the neutral sugar content of the CK and α-ZJP groups remained significantly decreased (p<0.05), but then decreased slowly. Throughout the fermentation period, the neutral sugar content in the PC and ZJP groups decreased significantly (p<0.05). After 48 h of fermentation, the reduction in neutral sugar content in the CK, PC, ZJP, and α-ZJP groups was 99.4%, 63.9%, 87.5%, and 94.2%, respectively. The consumption rate in the ZJP group was significantly higher than that in the PC group, with the α-ZJP group showing the fastest consumption. Although the initial sugar content in the ZJP and α-ZJP groups was lower than that in the PC group, the consumption was more complete (residual amount <0.3 mg / mL vs. 1.78 mg / mL in PC), which may have promoted the efficient utilization of sugar by microorganisms. The α-ZJP group had the lowest residual sugar content at 48 hours (0.11 mg / mL).

[0047] At 6 hours of fermentation, the uronic acid content in the α-ZJP group decreased from (1.20±0.02 mg / mL) to (0.35±0.01 mg / mL), representing 71% consumption (equivalent to 36.7% of the ZJP group). In the ZJP group, 81% consumption occurred at 12 hours, with the uronic acid content decreasing from (1.28±0.02 mg / mL) to (0.18±0.02 mg / mL). At 48 hours, uronic acid was almost completely consumed in all groups, with no significant difference in the remaining uronic acid content.

[0048] 3. SCFAs (the results are shown in Table 8) Table 8. Changes in SCFA levels at different fermentation time points. No SCFAs were detected in any group at 0 h. With prolonged culture time (6→12→24→48 h), the total SCFAs concentration significantly increased, indicating that prolonged culture time significantly promoted SCFAs accumulation, with the highest total SCFAs level observed in all groups at 48 h. At 6 h of fermentation, the α-ZJP group (16.75±0.83a) had significantly higher concentrations than ZJP (12.69±0.20b), PC (10.87±0.39c), and CK (9.12±0.46d), indicating that α-ZJP had the most significant effect on promoting SCFAs formation in the early stages. However, in subsequent fermentations, the ZJP group was significantly higher than the α-ZJP, PC, and CK groups, indicating that ZJP had the strongest ability to maintain SCFAs formation in the later stages. The α-ZJP group had the highest butyric acid yield (4.93 μmol / ml), with valeric acid synergistically accumulating (1.63 μmol / ml, 1.7 times that of the ZJP group). The ZJP group had significantly higher total SCFAs and acetic acid content, with acetic acid accounting for 61.4% (27.36 / 44.53). Compared with the CK and PC groups, the α-ZJP and ZJP groups were better utilized and metabolized by gut microbiota, indicating good prebiotic potential. Furthermore, the different fermentation advantages of the α-ZJP and ZJP groups suggest that α-galactosidase modification altered the original fermentation pathway of ZJP, effectively regulating gut microbiota to exert its prebiotic effect.

[0049] 4. Analysis of gut microbiota results Depend on Figure 13 As shown, after 48 hours of fermentation, the colony richness and diversity in the PC group significantly decreased, possibly due to the strong acidity inhibiting the growth of most colonies. The Chao1 index indicates that the ZJP group had the highest community richness, while the α-ZJP group had slightly lower richness than the CK group. The Simpson index shows that the community diversity of the ZJP and α-ZJP groups was higher than that of the CK and PC groups. The α-ZJP group had the highest community evenness, indicating reduced species competition and balanced resource allocation. In summary, the ZJP and α-ZJP groups maintained high community diversity and evenness, suggesting that these two treatments may be more conducive to maintaining community balance. The Beta diversity index focuses on comparing diversity between different growth environments, i.e., the differences between samples. Based on... Figure 14 Principal coordinate analysis (PCoA) showed that PC1 (67.7%) and PC2 (20.3%) contributed 86%, indicating that PCoA analysis can effectively explain >86% of the original information. The PC and CK groups clustered independently and were clearly separated from the other groups. The α-ZJP and ZJP groups were spatially close, indicating similar community structures, but not completely overlapping, and some differences existed. These results are consistent with data on α diversity, microbial composition, and SCFAs (superficial microbial products).

[0050] Figure 15 This indicates changes in gut microbiota at the phylum level among different groups. The dominant phyla in each group are Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes, accounting for >98% of all bacteria. In the CK group, the dominant phyla were Proteobacteria (44.52%), Firmicutes (43.72%), Bacteroidetes (6.00%), and Actinobacteria (4.63%), while in the PC group, the dominant phyla were Firmicutes (50.74%), Actinobacteria (26.13%), Proteobacteria (19.88%), and Bacteroidetes (3.21%). Actinobacteria are composed of some probiotics (such as Bifidobacteria), which can regulate the immune system, prevent intestinal diseases, control serum cholesterol levels, and fight cancer. After α-ZJP intervention, the abundance of Firmicutes (52.24%) and Actinobacteria (9.52%) was significantly increased compared to the CK group; after ZJP intervention, the abundance of Actinobacteria (12.21%) and Bacteroidetes (7.65%) was significantly increased compared to the CK group. The F / B ratio, which refers to the relative abundance of Firmicutes / Bacteroidetes, is one of the core indicators for assessing the structure and metabolic function of the gut microbiota. The ratios of the PC (15.81) and α-ZJP (13.26) groups were significantly higher than those of the CK (7.29) and ZJP (5.98) groups. The Proteobacteria phylum, composed of various pathogenic bacteria, shows an increase in abundance that is always accompanied by a decrease in the abundance of butyrate-producing bacteria. It is also increased in intestinal diseases such as colorectal cancer, diverticulitis, inflammatory bowel disease, type 2 diabetes, and obesity. After intervention with ZJP and α-ZJP, the abundance of Proteobacteria (33.99% and 33.61%, respectively) was significantly reduced compared to the control group. Overall, both ZJP and α-ZJP can effectively regulate the composition and abundance of the gut microbiota by increasing the proliferation of probiotics and reducing the proliferation of harmful bacteria.

[0051] Figure 16This indicates changes in gut microbiota at the family level among different groups. In the CK group, the prominent bacteria at the family level were f_Enterobacteriaceae (42.87%), f_Veillonellacea (16.43%), f_Lachnospiraceae (14.81%), and f_Ruminococcaceae (5.50%). f_Enterobacteriaceae (Enterobacteriaceae) is a family belonging to the phylum Proteobacteria, class Gamma-Proteobacteria, and order Enterobacteriaceae. Some strains are pathogenic; for example, enteropathogenic Escherichia coli can cause diarrhea and other diseases. Compared with the CK group, the abundance of this bacterium was significantly reduced in the ZJP (33.50%) and α-ZJP (32.74%) groups. f_Veillonellaceae can produce propionic acid to enhance intestinal barrier function and cooperate with Bacteroides and Clostridium in the intestine to maintain acidic metabolite balance and prevent sudden pH drops. Compared to the CK group, the abundance of f_Veillonellaceae was significantly increased in the ZJP (25.06%) and α-ZJP (25.55%) groups; the α-ZJP (18.79%) group had the highest abundance of f_Lachnospiraceae, which belongs to the Firmicutes phylum and is closely related to the host's health, metabolism, and immune function. It is adept at breaking down complex carbohydrates (such as dietary fiber) and producing short-chain fatty acids (SCFAs) through fermentation, such as butyric acid and propionic acid. Its abundance is positively correlated with gut health and may reduce the risk of inflammatory bowel disease (IBD), obesity, metabolic syndrome, and other diseases. f_Coriobacteriaceae is adept at utilizing carbohydrates that are difficult to digest directly (such as mucin and certain polysaccharides) and amino acids for metabolism, producing short-chain fatty acids (such as propionic acid), ammonia, and other metabolites, and is closely related to the host's metabolism, immunity, and disease status. The abundance of this bacterium in the ZJP (9.553%) and α-ZJP (6.91%) groups was significantly higher than that in the CK (3.37%) and PC (3.77%) groups. Bifidobacteria, as a physiologically beneficial bacterium, plays a variety of important physiological roles in human health, including biological barrier function, nutritional function, anti-tumor function, immune enhancement, improvement of gastrointestinal function, and anti-aging. The abundance of f_Bifidobacteriaceae in the ZJP (2.67%) and α-ZJP (2.60%) groups was more than twice that in the CK (1.24%) group. In conclusion, the polysaccharide groups exhibited different changes in the composition of the gut microbiota, but all produced beneficial effects, and can serve as potential prebiotics to regulate human intestinal function.

[0052] Figure 17 The heatmap analysis of gut microbiota abundance at the scientific level shows that the microbial composition of ZJP and α-ZJP groups is more similar but different, indicating that the modification of ZJP by α-galactosidase has a role in the regulation of gut microbiota.

[0053] from Figure 18 The Venn diagram shows the number of species unique to α-ZJP (734), ZJP (1116), PC (343), and CK (992). The number of species indicates different trends in species diversity among the polysaccharide groups. Figure 19 Cladogram and LDA score Figure 20 LEfSe analysis also revealed 8, 7, 10, and 5 distinct gut microbiota in the CK, PC, ZJP, and α-ZJP groups, respectively, with significant differences between groups. At the genus level, the CK group mainly exhibited high abundance of *g_Escherichia* and *g_Oscillospira*. The dominant bacteria in the PC group were *g_Megamona* and *g_Bifidobacterium*, while the dominant genera in the ZJP group were *g_Bacteroides*, *g_Clostridium f_Clostridiaceae*, and *g_Phascolarctobacterium*, and the dominant genera in the α-ZJP group were *g_Dialister* and *g_Faecalibacterium*. These findings suggest that ZJP and α-ZJP treatments have a significant impact on gut microbiota composition, potentially supporting gut microbiota health by promoting the growth of beneficial probiotics and inhibiting harmful bacteria.

[0054] The embodiments of this application have been described above with reference to the accompanying drawings. Specific examples have been used to illustrate the principles and implementation methods of this application. The description of the above embodiments is only for the purpose of helping to understand the core ideas of this application. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A modified polysaccharide for jujubes, characterized in that, The modified jujube polysaccharide contains 34.77 mg ± 0.504 mg of total sugar, 32.00 mg ± 2.01 mg of uronic acid, and 8.89 mg ± 0.22 mg of reducing sugar per 100 mg.

2. The jujube-modified polysaccharide according to claim 1, characterized in that, The monosaccharides contained in the modified jujube polysaccharide, by mole percentage, include the following: Fructose 0.37%, rhamnose 4.69%, arabinose 10.83%, galactose 11.90%, gluconic acid 2.12%, xylose 1.24%, mannose 0.81%, galacturonic acid 66.97%, glucuronic acid 1.07%.

3. The jujube-modified polysaccharide according to claim 2, characterized in that, The molecular weight of the modified jujube polysaccharide is (0.322±0.001)×10 4 Da - (215.835 ± 0.363) × 10 4 Da.

4. The method for preparing the modified jujube polysaccharide according to any one of claims 1-3, characterized in that, Includes the following steps: Jujube polysaccharide was placed in α-galactosidase solution, the pH was adjusted to 5.5, and the reaction was carried out at 37℃ for 1 h to inactivate it. After centrifugation, the supernatant was collected, precipitated with alcohol, reconstituted, and freeze-dried to obtain the jujube-modified polysaccharide.

5. The preparation method according to claim 4, characterized in that, The jujube polysaccharide was prepared by the following process: S1. Dissolve the jujube powder in a eutectic solvent, then extract it using ultrasound. After extraction, centrifuge to obtain the supernatant. S2. Add the supernatant to ethanol for alcohol precipitation, let it stand overnight at 2℃-4℃, and centrifuge after standing to obtain the precipitate. S3. The precipitate is reconstituted with water, then the protein is removed using Sevage reagent, dialyzed, and freeze-dried to obtain the jujube polysaccharide.

6. The preparation method according to claim 5, characterized in that, The ratio of jujube powder to eutectic solvent is 1:

25.

7. The preparation method according to claim 6, characterized in that, The eutectic solvent is obtained by dissolving choline chloride and anhydrous citric acid in water, wherein the molar ratio of choline chloride to anhydrous citric acid is 1:

1.

8. The preparation method according to claim 5, characterized in that, The Sevage reagent is a mixed solution of chloroform and n-butanol in a volume ratio of 4:

1.

9. The preparation method according to claim 5, characterized in that, The extraction power during ultrasonic extraction is 150W, the extraction temperature is 60℃-80℃, and the extraction time is 30min-40min.

10. The use of the modified jujube polysaccharide according to any one of claims 1-3 in the preparation of prebiotic foods that regulate intestinal flora.

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