Preparation method of degraded polysaccharide of griffithia sp. capable of regulating short-chain fatty acid level and application thereof

Abstract

The application discloses a preparation method of a degraded polysaccharide of Gracilaria lemaneiformis capable of regulating short-chain fatty acid level and application thereof, and belongs to the technical field of biotechnology. The method comprises the following steps: mixing Gracilaria lemaneiformis powder with water, performing ultrasonic cell wall disruption and hot water extraction, and obtaining a crude polysaccharide extract; adding cellulase and vitamin C to the crude polysaccharide extract, and performing synergistic degradation under ultrasonic conditions; after termination of the reaction, performing concentration, alcohol precipitation, gel column purification, dialysis and freeze-drying, and obtaining the degraded polysaccharide. The application firstly adopts ultrasonic assistance, cellulase and vitamin C to synergistically degrade Gracilaria lemaneiformis polysaccharide, and clearly shows that the degraded polysaccharide is composed of agarose typical structural units and red algal starch structural units. Animal experiments show that the degraded polysaccharide can significantly increase the total short-chain fatty acid content in the intestines of high-fat and high-sugar diet mice. The preparation method is mild, the structure is clear, the activity is significant, and the degraded polysaccharide can be applied to functional food or probiotic preparations for regulating intestinal flora and improving sugar and lipid metabolism.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for preparing and applying a short-chain fatty acid-modifying polysaccharide derived from Asparagus cochinchinensis. Background Technology

[0002] Seaweed (Alternanthera philoxeroides) is a traditional edible seaweed in my country, rich in polysaccharides and proteins, among other bioactive nutrients. It plays a role in regulating glucose and lipid metabolism, gut microbiota, and short-chain fatty acids, and can be used to develop dietary supplements, low-sugar foods, and metabolic regulation products, thus contributing to industry development. Short-chain fatty acids (SCFAs) are important metabolites produced by the fermentation of undigested carbohydrates by gut microbiota, playing a multifaceted physiological role in maintaining host health. Recent studies have shown that polysaccharides can regulate glucose and lipid metabolism and increase prebiotics by modulating gut microbiota and short-chain fatty acids. The relationship between seaweed polysaccharides and gut health has received increasing attention in recent years.

[0003] However, the natural polysaccharides from Gracilaria have structural defects such as large molecular weight, high viscosity, and poor solubility, which severely limit their full potential for biological activity and practical application. Currently, the degradation methods and biological activities of Gracilaria polysaccharides are key research directions. Reported degradation methods include physical methods such as ultrasound and microwave, biological methods such as fermentation and enzymatic hydrolysis, and chemical methods such as acid hydrolysis, oxidants, and reducing agents. The reported biological activities mainly include antioxidant, anti-inflammatory, hypoglycemic, hypolipidemic, immunomodulatory, and antitumor activities. For example, Guangdong Pharmaceutical University disclosed a method for extracting antioxidant substances from Gracilaria using ultrasound (CN116120481A). The Third Institute of Oceanography, Ministry of Natural Resources, disclosed a method for preparing and applying probiotic-fermented Gracilaria polysaccharides with multiple activities (CN120796412A), obtaining Gracilaria polysaccharides through co-culturing *Lactobacillus plantarum* HJ-S2 with Gracilaria. South China University of Technology has disclosed a Gracilaria polysaccharide with significant anti-inflammatory activity, its preparation method, and its application (CN112961260A). The Gracilaria polysaccharide was prepared using H2O2 and ultraviolet irradiation. They also disclosed a method for preparing functional oligosaccharides from Gracilaria using water extraction, which can promote the growth and proliferation of probiotics and produce acid (CN110128561A). Furthermore, they disclosed a Gracilaria polysaccharide with significant lipid-lowering activity, its preparation method, and its application (CN108586632A), prepared using pre-homogenization, organic acid combined with dynamic high-pressure microfluidic technology. Dalian University of Technology has disclosed a method for preparing Gracilaria polysaccharide oligosaccharides and its application (CN110368400A), using water extraction, alcohol precipitation, and acid hydrolysis to prepare oligosaccharides, and studying the composition and expression of intestinal flora and short-chain fatty acids. The South China Sea Fisheries Research Institute of the Chinese Academy of Fishery Sciences has disclosed a high-purity, low-molecular-weight polysaccharide from *Gracilaria fusiforme* with antioxidant activity, its preparation method, and its application (CN115093490A). The low-molecular-weight polysaccharide is prepared using H2O2 and vitamin C. The aforementioned patents mainly focus on the extraction and bioactivity of *Gracilaria fusiforme* polysaccharides, lacking reports on the structure of the polysaccharides or oligosaccharides. Currently, there are no literature reports on the preparation of *Gracilaria fusiforme* degraded polysaccharides using a combination of ultrasound, cellulase, and vitamin C, nor on the determination of their regulatory effects on short-chain fatty acids. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing and applying *Asparagus cochinchinensis* degraded polysaccharides with adjustable short-chain fatty acid levels, thereby addressing the problems existing in the prior art. This invention utilizes the synergistic degradation of short-chain fatty acids by ultrasound, cellulase, and vitamin C under mild conditions to obtain low-molecular-weight degraded polysaccharides, and uses multidimensional nuclear magnetic resonance (NMR) to clarify their repeating structural units. These degraded polysaccharides can significantly increase the content of total short-chain fatty acids, acetic acid, propionic acid, butyric acid, isobutyric acid, and isovaleric acid in the intestines of mice on a high-fat, high-sugar diet. They can be used as functional foods or probiotic preparations to regulate intestinal flora and improve glucose and lipid metabolism.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a degradable polysaccharide from Gracilaria fusiforme, which comprises agarose structural units and red algae starch structural units, wherein the chemical structure of the agarose structural unit is as follows: ; The chemical structure of the red algae starch structural unit is as follows: .

[0006] The present invention also provides a method for preparing the aforementioned Gracilaria degraded polysaccharide, comprising the following steps: (1) After dissolving the asparagus powder in water, ultrasonically break it up, and then extract it by shaking in hot water to obtain a crude polysaccharide extract. (2) Add cellulase and vitamin C to the crude polysaccharide extract and carry out the degradation reaction under ultrasonic conditions to obtain the degraded crude polysaccharide extract; (3) Terminate the degradation reaction and concentrate the crude extract of the degraded polysaccharide to obtain a concentrated degraded polysaccharide solution; (4) Add anhydrous ethanol to the concentrated degraded polysaccharide solution for alcohol precipitation, and then purify by gel column, dialyze and freeze dry to obtain the degraded polysaccharide of Asparagus cochinchinensis.

[0007] Preferably, in step (1), the ratio of the asparagus powder to water is 1:25 to 1:60; The ultrasonic cell disruption process has a power of 300-600 W, a temperature of 20-40℃, and a time of 20-60 min. The temperature for extraction by shaking in hot water is 70~90℃, and the time is 2~4 h.

[0008] Preferably, in step (2), the final concentration of the cellulase is 0.5% to 2%, and the final concentration of the vitamin C is 0.05 to 2 mol / L.

[0009] Preferably, in step (2), the conditions for the degradation reaction are: ultrasonic power of 300~600 W, temperature of 40~65℃, and time of 0.5~1 h.

[0010] Preferably, in step (4), the amount of anhydrous ethanol added is 3 to 5 times that of the degraded polysaccharide concentrate.

[0011] Preferably, in step (4), the gel column purification is performed using glucose gel G-100 with gradient elution using 0-1 M sodium chloride solution as the eluent, and the elution flow rate is 0.5-2 mL / min.

[0012] Preferably, in step (4), the 0 M sodium chloride solution eluent is dialyzed for 48 to 96 h using a dialysis bag with a molecular weight cutoff of 5000 to 10000 Da.

[0013] This invention also provides the application of the aforementioned Gracilaria degraded polysaccharide in any of the following: (1) Application in the preparation of products that regulate intestinal short-chain fatty acids; (2) Application in the preparation of products that regulate intestinal flora and improve glucose and lipid metabolism.

[0014] The present invention also provides a product comprising the aforementioned Asparagus polysaccharide.

[0015] The present invention discloses the following technical effects: This invention is the first to employ ultrasound-assisted degradation of Gracilaria polysaccharide using a synergistic combination of cellulase and vitamin C. This approach effectively reduces molecular weight, improves solubility and bioavailability under mild conditions, and avoids the damage to the active structure caused by traditional acid hydrolysis. Through methylation and multidimensional NMR analysis, it was first clarified that the degraded polysaccharide consists of the typical agarose structural unit (→3)- β -D-Gal p -(1→4)- α -3,6-anhydro-D-Gal p -(1→) and red algal starch structural unit (→4)- α -D-Glc p The repeating structure is characterized by -(1→). Animal experiments have shown that this degraded polysaccharide can significantly increase the total short-chain fatty acid content in the intestines of mice on a high-fat, high-sugar diet. In particular, the effect on increasing acetic acid, propionic acid, butyric acid, isobutyric acid, and isovaleric acid is significantly better than that of undegraded polysaccharide. It can be applied to functional foods or probiotic preparations that regulate intestinal flora and improve glucose and lipid metabolism. Attached Figure Description

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

[0017] Figure 1 For the degradation of polysaccharides 1 H NMR spectrum; Figure 2 For the degradation of polysaccharides 13 C NMR spectrum; Figure 3 For the degradation of polysaccharides 1 H-1 H COSY spectrum; Figure 4 For the degradation of polysaccharides 1 H- 13 C HSQC spectrum; Figure 5 For the degradation of polysaccharides 1 H- 13 C HMBC spectrum; Figure 6 For the degradation of polysaccharides 1 H- 1 H NOESY spectrum; Figure 7 These are the constituent and repeating structural units of polysaccharides; Figure 8 The regulation of short-chain fatty acids in mice by the degradation of polysaccharides from Asparagus officinalis; A: total short-chain fatty acids, B: acetic acid, C: propionic acid, D: butyric acid; Figure 9 The regulation of short-chain fatty acids in mice by the degradation of polysaccharides from Asparagus officinalis; A: valeric acid, B: isobutyric acid, C: isovaleric acid, D: hexanoic acid; The above Figure 8 and Figure 9 In the study, GLP-HV represented degraded polysaccharides, while GLP represented undegraded polysaccharides as a control. "#" indicates a significant difference from the NC group (P < 0.05), and "*" indicates a significant difference from the MODE group (P < 0.05). Detailed Implementation

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

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

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

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

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

[0023] Example 1: Preparation of degraded polysaccharides Take an appropriate amount of Asparagus cochinchinensis powder and add pure water at a ratio of 1:25 (w / v). First, ultrasonically break the cell wall for 20 min at a power of 450 W and a temperature of 30℃. Then, extract with hot water shaking (150 rpm) at 70℃ for 4 h to obtain crude polysaccharide extract.

[0024] Cellulase and vitamin C were added to the crude polysaccharide extract and the mixture was degraded under ultrasonic conditions at 300 W and 40 °C for 1 h. The final concentration of cellulase was 0.5% and the final concentration of vitamin C was 0.05 mol / L.

[0025] Add 1 M sodium hydroxide to adjust the pH to 7.0 to terminate the vitamin C degradation reaction. Then, inactivate the enzyme in 100 °C hot water for 10 min to end the degradation reaction. After cooling, concentrate the crude polysaccharide extract at 60 °C using a vacuum rotary evaporator to obtain the concentrated polysaccharide extract.

[0026] Add 4 volumes of anhydrous ethanol to the concentrated degraded polysaccharide solution, and purify using a glucose gel G-100 column at the following conditions: column size (1.6 cm × 70 cm), NaCl solution as the eluent, and elute as follows: The NaCl concentration is 0M (i.e., the eluent is water), 50 tubes are collected, the elution flow rate is 1 mL / min, and 5 mL is collected from each tube.

[0027] The NaCl concentration was 0.2 M, 50 tubes were collected, the elution flow rate was 1 mL / min, and 5 mL was collected from each tube.

[0028] The NaCl concentration was 0.4 M, 50 tubes were collected, the elution flow rate was 1 mL / min, and 5 mL was collected from each tube.

[0029] The NaCl concentration was 0.6 M, 50 tubes were collected, the elution flow rate was 1 mL / min, and 5 mL was collected from each tube.

[0030] The NaCl concentration was 0.8 M, 50 tubes were collected, the elution flow rate was 1 mL / min, and 5 mL was collected from each tube.

[0031] The NaCl concentration was 1 M, and 50 tubes were collected. The elution flow rate was 1 mL / min, and 5 mL was collected from each tube.

[0032] The polysaccharide content was determined using the conventional phenol-sulfuric acid method. The final eluent containing polysaccharides was collected when eluting with water at a NaCl concentration of 0M. The eluents containing polysaccharides were combined and dialyzed with a 7000 Da dialysis bag for 72 h (with water changed 3 times a day) and then freeze-dried under vacuum to prepare the purified degraded polysaccharide, named GLP-HV.

[0033] The formula for calculating total sugar is: In the above formula, c is calculated from the standard curve, v is the total volume, m is the mass of the raw material, and 0.9 is the conversion coefficient between polysaccharides and reducing sugars.

[0034] Example 2 Preparation of degraded polysaccharides Take an appropriate amount of Asparagus cochinchinensis powder and add pure water at a ratio of 1:30 (w / v). First, ultrasonically break the cell wall for 40 min at a power of 450 W and a temperature of 30℃. Then, extract with hot water shaking (150 rpm) at 80℃ for 3 h to obtain crude polysaccharide extract.

[0035] Cellulase and vitamin C were added to the crude polysaccharide extract and subjected to ultrasonic degradation at 500 W and 50 °C for 40 min. The final concentration of cellulase was 1% and the final concentration of vitamin C was 1 mol / L.

[0036] Add 1 M sodium hydroxide to adjust the pH to 7.0 to terminate the vitamin C degradation reaction. Then, inactivate the enzyme in 100℃ hot water for 10 min to end the degradation reaction. After cooling, concentrate the crude extract of degraded polysaccharides at 60℃ using a vacuum rotary evaporator to obtain the concentrated degraded polysaccharide solution.

[0037] Add 4 volumes of anhydrous ethanol to the concentrated degraded polysaccharide solution, and purify using a glucose gel G-100 column at the following conditions: column size (1.6 cm × 70 cm), NaCl solution as the eluent, and elute as follows: The NaCl concentration is 0M (i.e., the eluent is water), 50 tubes are collected, the elution flow rate is 1 mL / min, and 5 mL is collected from each tube.

[0038] The NaCl concentration was 0.2 M, 50 tubes were collected, the elution flow rate was 1 mL / min, and 5 mL was collected from each tube.

[0039] The NaCl concentration was 0.4 M, 50 tubes were collected, the elution flow rate was 1 mL / min, and 5 mL was collected from each tube.

[0040] The NaCl concentration was 0.6 M, 50 tubes were collected, the elution flow rate was 1 mL / min, and 5 mL was collected from each tube.

[0041] The NaCl concentration was 0.8 M, 50 tubes were collected, the elution flow rate was 1 mL / min, and 5 mL was collected from each tube.

[0042] The NaCl concentration was 1 M, and 50 tubes were collected. The elution flow rate was 1 mL / min, and 5 mL was collected from each tube.

[0043] The polysaccharide content was determined using the conventional phenol-sulfuric acid method. The final eluent containing polysaccharides was collected when eluting with water at a NaCl concentration of 0M. The eluents containing polysaccharides were combined and dialyzed with a 7000 Da dialysis bag for 72 h (with water changed 3 times a day) and then freeze-dried under vacuum to prepare the purified degraded polysaccharide, named GLP-HV.

[0044] Example 3 Preparation of degraded polysaccharides Take an appropriate amount of Asparagus cochinchinensis powder and add pure water at a ratio of 1:50 (w / v). First, ultrasonically break down the cell walls for 60 min at a power of 450 W and a temperature of 30℃. Then, extract the polysaccharide crude extract by hot water shaking (150 rpm) at 90℃ for 2 h.

[0045] Cellulase and vitamin C were added to the crude polysaccharide extract and subjected to ultrasonic degradation at 500 W and 60 °C for 0.5 h. The final concentration of cellulase was 2% and the final concentration of vitamin C was 2 mol / L.

[0046] Add 1 M sodium hydroxide to adjust the pH to 7.0 to terminate the vitamin C degradation reaction. Then, inactivate the enzyme in 100℃ hot water for 10 min to end the degradation reaction. After cooling, concentrate the crude extract of degraded polysaccharides at 60℃ using a vacuum rotary evaporator to obtain the concentrated degraded polysaccharide solution.

[0047] Add 4 volumes of anhydrous ethanol to the concentrated degraded polysaccharide solution, and purify using a glucose gel G-100 column at the following conditions: column size (1.6 cm × 70 cm), NaCl solution as the eluent, and elute as follows: The NaCl concentration is 0M (i.e., the eluent is water), 50 tubes are collected, the elution flow rate is 1 mL / min, and 5 mL is collected from each tube.

[0048] The NaCl concentration was 0.2 M, 50 tubes were collected, the elution flow rate was 1 mL / min, and 5 mL was collected from each tube.

[0049] The NaCl concentration was 0.4 M, 50 tubes were collected, the elution flow rate was 1 mL / min, and 5 mL was collected from each tube.

[0050] The NaCl concentration was 0.6 M, 50 tubes were collected, the elution flow rate was 1 mL / min, and 5 mL was collected from each tube.

[0051] The NaCl concentration was 0.8 M, 50 tubes were collected, the elution flow rate was 1 mL / min, and 5 mL was collected from each tube.

[0052] The NaCl concentration was 1 M, and 50 tubes were collected. The elution flow rate was 1 mL / min, and 5 mL was collected from each tube.

[0053] The polysaccharide content was determined using the conventional phenol-sulfuric acid method. The final eluent containing polysaccharides was collected when eluting with water at a NaCl concentration of 0M. The eluents containing polysaccharides were combined and dialyzed with a 7000 Da dialysis bag for 72 h (with water changed 3 times a day) and then freeze-dried under vacuum to prepare the purified degraded polysaccharide, named GLP-HV.

[0054] Example 4: Structural Analysis of Degraded Polysaccharides The following detection and analysis were performed using the degraded polysaccharide GLP-HV prepared in Example 2 as an example.

[0055] 1. Methylation analysis and mass spectrometry analysis Polysaccharide methylation analysis was performed using gas chromatography-mass spectrometry (GC-MS). The chromatographic parameters were as follows: an Agilent 7820A gas chromatograph was used, with a BPX70 column (30 m × 0.25 mm × 0.25 µm) selected based on the compound's properties. The injection volume was 1 μL, the split ratio was 10:1, and high-purity helium was used as the carrier gas at a flow rate of 1 mL / min. The column oven temperature was initially set at 140 °C for 2 min, then programmed to increase to 230 °C at a rate of 3 °C / min and held for 3 min. Mass spectrometry parameters were as follows: an Agilent 5977B quadrupole mass spectrometer was used. An electron impact ion source was employed, and the analyte was detected in full scan mode. The optimized mass spectrometry conditions were as follows: injection port temperature 230 °C, quadrupole temperature 150 °C, full scan mode, and mass scan range (m / z): 30-600.

[0056] 2. Nuclear magnetic resonance analysis One-dimensional analysis of polysaccharides was performed using a 600 MHz nuclear magnetic resonance spectroscopy instrument. 1 H spectrum and 13 C-spectrum) and two-dimensional NMR analysis (COSY, HSQC, HMBC and NOESY), with a scanning temperature of 25 °C.

[0057] 3. Results and Analysis 3.1 Results of methylation analysis The methylated sample fragments were compared and analyzed with a standard sugar residue-derived PMAAs mass spectrometry database. The methylated GC-MS results were analyzed to obtain various parameters and proportions for each monosaccharide residue. The results showed that glucose was mainly linked by four glycosidic bonds: tD-Glcp, 1,4-D-Glcp, 1,3,4-D-Glcp, and 1,4,6-D-Glcp. Galactose was mainly linked by four glycosidic bonds: tD-Galp, 1,3-D-Galp, 1,4,6-D-Galp, and 1,3,6-D-Galp. Based on the analysis of monosaccharides and the methylation results, it can be preliminarily determined that the chemical structure of GLP-HV is likely mainly composed of galactose linked by 1,3 glycosidic bonds and glucose linked by 1,4 glycosidic bonds. Further analysis using NMR spectroscopy will determine the presence of glycosidic bonds and possible substituents.

[0058] 3.2 NMR Analysis Results The above methylation analysis has preliminarily determined the type and linkage mode of glycosidic bonds. Further analysis using one-dimensional and two-dimensional NMR spectra can further confirm and verify the linkage mode and sequence of glycosidic bonds. The NMR spectra are shown below. Figures 1-6 As shown.

[0059] In the proton NMR spectrum1 In 1H NMR, the anomeric hydrogen proton H1 appears in the region of δ 4.3–6.0 ppm. α The anoprotons of type glycosidic bonds are generally in the range of δ 5.0-6.0 ppm, while β The anoprotons of type glycosidic bonds are generally in the range of δ 4.3-5.0 ppm. For example... Figure 1 As shown, excluding the strongest solvent peak at 4.70 ppm (a heavy water proton signal), this polysaccharide exhibits multiple anomeric H1 proton signals in the δ 4.3–6.0 ppm range. Combined with methylation information, information near the main signals δ 5.32, 5.21, 5.07, 4.58, and 4.48 ppm in the spectrum was identified (see Table 1). The results indicate that the majority of glycosidic bonds in the structure are of the methylation type 5.32–6.0 ppm. α Constructive residues, a small portion of which are present β Configuration residues. In carbon NMR spectroscopy. 13 In C NMR, such as Figure 2 As shown, the anomeric carbon C1 resonance signal of polysaccharides generally appears in the range of δ 90-112 ppm. The main anomeric carbon signals are identified at positions around δ 97.93, 99.72, 100.78, 101.82, and 102.88 ppm. Methylation determined the main residue composition of the monosaccharides. A comprehensive analysis and assignment of the NMR signals yielded the NMR H / C position values ​​for various sugar residues in Table 1. Through careful analysis of the NMR spectra, combined with the methylation results, and based on the typical structural characteristics of polysaccharides in *Gynostemma pentaphyllum*, the presence of a dehydrated A residue was identified. This residue represents a characteristic structure formed by the condensation and dehydration of galactose at positions 3 and 6. While no corresponding PMAA data was found in the methylation results, a clear signal appeared in the NMR, leading to the determination of the structure.

[0060] Table 1. NMR values ​​of various sugar residues 1 H / 13 Chemical shift of C like Figures 3-6 As shown in Table 2, by analyzing the COSY, HSQC, HMBC, and NOESY spectra, the glycosidic bond sequence between sugar residues can be determined, and the possible structural units can be deduced from this, as shown in Table 2. Furthermore, the main chain structure and side chain composition of the polysaccharide can be analyzed. First, a strong cross-peak signal at δ 5.04 / 81.89 ppm was found in the spectra. Based on the above assignment signals, this indicates the presence of 1,4- α -3,6-anhydro-D-Gal p →3- β -D-Gal p-1→ Such a structural unit, and then finding a strong cross-peak signal of δ 4.471 / 79.47 ppm, based on the above attribution signal, indicates the existence of →3- β -D-Gal p- 1→4- α -3,6-anhydro-D-Gal p- 1 → This structural unit; these two signals suggest that the galactose in this polysaccharide contains relatively obvious repeating links of A and C residues, constituting a typical structural unit of agarose. It is worth noting that small amounts of 4,6-Gal and T-Gal residues can also be found during methylation. Combining the above signal attributions with the signals in the two-dimensional NMR, it is inferred that these are small branched structures connected within the agarose backbone, characterized by backbone connections formed at position 4 of the residues and small amounts of short branched structures formed at position 6.

[0061] The monosaccharide composition and methylation test results showed a high glucose content. Based on the residue assignments in Table 1 and the strong δ signals of 5.28 / 3.55 ppm in Table 2, it was determined that →4- was present in the main chain. α -D-Glc p -1→4- α -D-Glc p- 1→ Such structural units constitute the glycosidic bonds between glucose monosaccharides; based on the δ 4.88 / 3.85 ppm signal, the less abundant glucose residues 1,4,6- α -D-Glc p It may form 1→6 glycosidic bonds in the side chain by linking with the same monosaccharide; based on the two-dimensional NMR spectrum and residue structure characteristics, it is determined that the glucose in this polysaccharide constitutes a typical red algae starch structure.

[0062] Table 2. Glycosidic bond analysis of two-dimensional NMR spectra Therefore, based on the overall analysis above, it is inferred that the polysaccharide structure consists of two main structural units. One unit is a typical agarose unit, characterized by the 3-Gal residues forming repeating connections with dehydrated structures to form the structure. The other unit is a red algal starch structure, characterized by 4-Glc residues as the main sugar residues, with a small number of side chains forming at the 6-position. Therefore, possible repeating structural units include... Figure 7 As shown.

[0063] animal experiments 1. Experimental Materials Kunming (KM) male mice (14-16 g, 4 weeks old), feed and bedding were purchased from Guangdong Provincial Laboratory Animal Center (SCXK20220002, Foshan, Guangdong, China).

[0064] 2. Experimental Methods KM mice were acclimatized in an animal facility for 7 days (temperature 22±2 ℃, relative humidity 50±5%), with ample sterile water and maintenance feed provided, and bedding changed every 2 days. Mice were randomly divided into 5 groups (n=10 per group): normal group (NC), model group (MODE), positive group (MET, metformin), GLP experimental group (following the experimental method of Example 2, excluding the steps of adding vitamin enzymes and vitamin C for degradation, otherwise the same as Example 2), and GLP-HV experimental group (GLP-HV prepared in Example 2). The dosage of both drugs and samples was 200 mg / (kg·bw) / day, administered by gavage once a day, with free access to feed and drinking water. The NC group was fed SPF-grade feed, while the other groups were fed a high-fat, high-sugar diet. After 8 weeks, mice were sacrificed according to animal ethics requirements, and intestinal contents were immediately removed for GC-MS analysis of intestinal short-chain fatty acids.

[0065] 3. Results and Analysis Short-chain fatty acids (SCFAs) are primarily produced by beneficial bacteria in the colon through the fermentation of indigestible carbohydrates. They are the main energy source for intestinal epithelial cells and play a crucial role in regulating gut microbiota and improving intestinal function, thus being closely related to glucose and lipid metabolism. Treatment of high-fat, high-glucose mice with the degraded polysaccharide GLP-HV prepared in this invention, as shown... Figure 8 As shown in Figure A, the total SCFAs contents of the NC group, MODE group, GLP group, and GLP-HV group were 878.05, 624.75, 615.81, and 866.67 µg / g, respectively. Figure 8 Chinese BD and Figure 9 As shown in the AD diagram, compared with the MODE group, the GLP-HV group showed significantly increased levels of acetic acid, propionic acid, butyric acid, isobutyric acid, and isovaleric acid. However, there was no significant difference in the levels of valeric acid and hexanoic acid between the GLP and MODE groups. Beneficial SCFA metabolites such as acetic acid, propionic acid, and butyric acid can improve insulin sensitivity and reduce lipid accumulation. Acetic acid plays a crucial role in promoting fatty browning and regulating cholesterol metabolism. Propionic acid can inhibit the activity of HMG-CoA reductase, thereby helping to lower serum cholesterol levels. Butyric acid plays a key role in enhancing insulin sensitivity, is a major energy source for intestinal epithelial cells, promotes the differentiation of regulatory T cells, and inhibits the production of pro-inflammatory cytokines. SCFAs are an important link between the gut microbiota and host metabolic health, and have the potential to significantly improve insulin sensitivity and regulate glucose and lipid metabolism.

[0066] Application examples The prepared Gracilaria lemaneiformis degrading polysaccharide can be applied to probiotic powder, increase the content of short-chain fatty acids, and regulate intestinal flora. For example, the degrading polysaccharide is mixed with fructo-oligosaccharide at a mass ratio of 1:1, and is added to probiotic powder, and the recommended daily intake is 5 g.

[0067] The above-described embodiments are only used to describe the preferred modes of the present application, and are not used to limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope of the present application defined by the claims.

Claims

1. A type of degraded polysaccharide from Asparagus setaceus, characterized in that, The *Gracilaria rubra* degraded polysaccharide comprises agarose structural units and red algae starch structural units, wherein the chemical structure of the agarose structural unit is as follows: ； The chemical structure of the red algae starch structural unit is as follows: 。 2. A method for preparing the degraded polysaccharide from Gracilaria as described in claim 1, characterized in that, Includes the following steps: (1) After dissolving the asparagus powder in water, ultrasonically break it up, and then extract it by shaking in hot water to obtain a crude polysaccharide extract. (2) Add cellulase and vitamin C to the crude polysaccharide extract and carry out the degradation reaction under ultrasonic conditions to obtain the degraded crude polysaccharide extract; (3) Terminate the degradation reaction and concentrate the crude extract of the degraded polysaccharide to obtain a concentrated degraded polysaccharide solution; (4) Add anhydrous ethanol to the concentrated degraded polysaccharide solution for alcohol precipitation, and then purify by gel column, dialyze and freeze dry to obtain the degraded polysaccharide of Asparagus cochinchinensis.

3. The preparation method according to claim 2, characterized in that, In step (1), the ratio of the asparagus powder to water is 1:25 to 1:60; The ultrasonic cell disruption process has a power of 300-600 W, a temperature of 20-40℃, and a time of 20-60 min. The temperature for extraction by shaking in hot water is 70~90℃, and the time is 2~4 h.

4. The preparation method according to claim 2, characterized in that, In step (2), the final concentration of the cellulase is 0.5% to 2%, and the final concentration of the vitamin C is 0.05 to 2 mol / L.

5. The preparation method according to claim 2, characterized in that, In step (2), the conditions for the degradation reaction are: ultrasonic power of 300~600 W, temperature of 40~65℃, and time of 0.5~1 h.

6. The preparation method according to claim 2, characterized in that, In step (4), the amount of anhydrous ethanol added is 3 to 5 times that of the degraded polysaccharide concentrate.

7. The preparation method according to claim 2, characterized in that, In step (4), the gel column purification is performed using glucose gel G-100 with gradient elution using 0-1 M sodium chloride solution as the eluent at a flow rate of 0.5-2 mL / min.

8. The preparation method according to claim 2, characterized in that, In step (4), the 0 M sodium chloride solution eluent is dialyzed for 48 to 96 h using a dialysis bag with a molecular weight cutoff of 5000 to 10000 Da.

9. The application of the Gracilaria degraded polysaccharide as described in claim 1 in any of the following: (1) Application in the preparation of products that regulate intestinal short-chain fatty acids; (2) Application in the preparation of products that regulate intestinal flora and improve glucose and lipid metabolism.

10. A product characterized in that, Includes the Gracilaria polysaccharide as described in claim 1.

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