A kind of D-galacturonan homogeneous pectin polysaccharide and its preparation method and application

Abstract

The present invention relates to the technical field of natural product preparation, and specifically to a D-galacturonic acid polysaccharide homogeneous pectin polysaccharide and its preparation method and application. The monosaccharide composition of the polysaccharide includes galacturonic acid, galactose, rhamnose, glucuronic acid and glucose; its skeleton structure is composed of →4)-α-GalpA-(1→ and →4)-α-GalpA-6-O-methyl-(1→. The polysaccharide can be obtained by lactic acid bacteria fermentation and extraction of Liangping pomelo and purification by column chromatography. The preparation method is simple, safe and suitable for expanded production. The polysaccharide has α-glucosidase inhibitory activity, and the α-glucosidase inhibition rate can reach 80%. IC 50 The value can reach 0.128 mg / mL, which lays a foundation for further research on α-glucosidase inhibitors and drugs for treating diabetes. It has broad promotion and application value, and at the same time creates conditions for the comprehensive utilization of Liangping pomelo resources.

Description

Technical Field

[0001] The present invention relates to the technical field of natural product preparation, and in particular to a D-galacturonan homogeneous pectin polysaccharide and a preparation method and application thereof. Background Art

[0002] α-glucosidase inhibitors inhibit α-glucosidase in the brush border of the small intestinal mucosa, thereby delaying carbohydrate absorption and reducing postprandial hyperglycemia. Key features of existing α-glucosidase inhibitors include stable blood sugar reduction, high safety, and reduced incidence of cardiovascular complications. They are among the few oral hypoglycemic agents that can address impaired glucose tolerance. Commonly used α-glucosidase inhibitors are primarily acarbose and voglibose. Acarbose is used to treat diabetes or lower postprandial blood sugar in patients with impaired glucose tolerance. However, it is not suitable for patients allergic to acarbose, those with chronic gastrointestinal disorders with significant digestive and absorption disorders, or those with severe renal impairment. Voglibose is used to improve postprandial hyperglycemia in diabetes. However, it is not suitable for patients with severe ketosis, those in diabetic coma or pre-coma, those with severe infections, or those undergoing surgery. To overcome the shortcomings of existing α-glucosidase inhibitors, the development of new alternatives is urgently needed. my country is rich in natural product resources. Screening, separation and purification of α-glucosidase inhibitors from natural products are low-cost, abundant in sources, challenging and valuable to develop, and have become the direction of development of new α-glucosidase inhibitors.

[0003] Liangping pomelo (Citrus maxima (Burm.) Merr. cv. Liangping pomelo) is a plant of the genus Citrus in the family Rutaceae. It is one of the three most famous pomeloes in China and is mainly produced in Liangping District, Chongqing, with a long history of cultivation. Due to the lack of effective utilization methods, the utilization rate of pomelo peel is extremely low. Pomelo peel is rich in pectin, flavonoids, limonoid compounds, dietary fiber, naringin, essential oils and other active ingredients. However, there are no reports in the prior art on the α-glucosidase inhibitory function of Liangping pomelo extracts. If the composition and efficacy of pomelo peel extracts can be fully studied, if a natural α-glucosidase inhibitor can be discovered, the problem of the existing technology lacking natural α-glucosidase inhibitors can be overcome, and the medicinal value of Liangping pomelo can be enhanced, further fully utilizing pomelo peel resources and improving the comprehensive utilization efficiency of Liangping pomelo raw materials. Summary of the Invention

[0004] The present invention aims to provide a D-galacturonan homogeneous pectin polysaccharide to solve the technical problem of the lack of natural α-glucosidase inhibitors in the prior art.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A D-galacturonan homogeneous pectin polysaccharide, whose monosaccharide composition includes galacturonic acid, galactose, rhamnose, glucuronic acid and glucose; and its backbone structure is composed of →4)-α-GalpA-(1→ and →4)-α-GalpA-6-O-methyl-(1→).

[0007] The present technical solution also provides a method for preparing D-galacturonan homogeneous pectin polysaccharide, comprising the following steps performed in sequence:

[0008] S1 Preparation of crude polysaccharide: The white inner skin of pomelo peel is taken and crushed to obtain pomelo peel powder; water, carbon source, and bacterial strain are then added to obtain crude polysaccharide through fermentation;

[0009] S2 purification: After the crude polysaccharide was subjected to alcohol precipitation and deproteinization treatment, it was sequentially chromatographed on a DEAE-52 cellulose column, a G-100 dextran gel column, and a G-75 dextran gel column to obtain citronellal polysaccharide PPs-1Aa.

[0010] The present technical solution also provides a use of D-galacturonan homopectin polysaccharide in the preparation of drugs for inhibiting α-glucosidase or treating diabetes.

[0011] In summary, the beneficial effects of this technical solution are:

[0012] This technical solution uses a microbial fermentation method to extract crude Liangping pomelo polysaccharides. This is then purified through chromatography on a DEAE-52 cellulose column, a G-100 dextran gel column, and a G-75 dextran gel column to obtain a novel citrus peel polysaccharide, designated PPs-1Aa. PPs-1Aa exhibits superior α-glucosidase inhibitory activity to acarbose, making it a potential alternative to conventional α-glucosidase inhibitors and worthy of further research and promotion. The inventors further investigated the structural composition of PPs-1Aa and discovered that it is a citrus peel polysaccharide primarily composed of monosaccharide components: galacturonic acid, galactose, rhamnose, glucuronic acid, and glucose, including a polysaccharide backbone structure composed of →4)-α-GalpA-(1→) and →4)-α-GalpA-6-O-methyl-(1→).

[0013] There are no reports in the prior art regarding the α-glucosidase inhibitory activity of pomelo peel extracts. This solution discovered for the first time that pomelo peel contains polysaccharide components with α-glucosidase inhibitory activity, and separated, purified, and structurally identified them. This solution uses microbial fermentation to effectively improve the physical and chemical properties of plant polysaccharides. During the fermentation process, high-molecular substances can be decomposed into small molecules, which has the potential to produce new active substances, allowing the effective ingredients to play a better role and reduce toxic side effects. This technical solution obtains a new type of α-glucosidase inhibitor by combining microbial fermentation with traditional separation and purification, which is expected to be applied in the medical practice of treating diseases related to sugar metabolism.

[0014] Furthermore, the molar ratio of galacturonic acid, galactose, rhamnose, glucuronic acid, and glucose is 86.8:3.87:4.23:1.11:0.57.

[0015] Furthermore, a D-galacturonan homogeneous pectin polysaccharide, the residues and molar percentages obtained by methylation analysis are:

[0016] 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl galactitol 8.04%,

[0017] 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl galactitol 86.09%,

[0018] 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl glucitol 1.00%,

[0019] 1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl galactitol 0.88%,

[0020] 1,3,4,5-tetra-O-acetyl-2,6-di-O-methyl galactitol 1.91%,

[0021] 1,4,5,6-tetra-O-acetyl-2,3-di-O-methyl glucitol 2.07%.

[0022] Furthermore, the bonding structure of 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl galactitol is t-Gal(p)-UA;

[0023] The bonding structure of 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl galactitol is 4-Gal(p)-UA;

[0024] The bonding structure of 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl glucitol is 4-Glc(p);

[0025] The bonding structure of 1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl galactitol is 6-Gal(p);

[0026] The bonding structure of 1,3,4,5-tetra-O-acetyl-2,6-di-O-methyl galactitol is 3,4-Gal(p);

[0027] The bonding structure of 1,4,5,6-tetra-O-acetyl-2,3-di-O-methyl glucitol is 4,6-Glc(p)-UA.

[0028] Furthermore, its molecular weight is 42.8 kDa.

[0029] Furthermore, the IC value of its inhibitory effect on α-glucosidase is 50 The value is 0.128mg / mL.

[0030] Furthermore, in S1, the fermentation conditions are: material-liquid ratio 20.5 mL / g, inoculation amount 10% of the mass of pomelo peel powder, fermentation time 62.5 h, and fermentation temperature 41°C.

[0031] Furthermore, in S1, the carbon source includes skim milk powder at 2% by weight of pomelo peel powder and white sugar at 1.5% by weight of pomelo peel powder; the bacterial strains are composed of Lactobacillus bulgaricus and Streptococcus thermophilus. Lactic acid bacteria are a general term for Gram-positive bacteria that ferment carbohydrates and produce lactic acid. They have multiple functions, including antibacterial, maintaining intestinal balance, promoting nutrient absorption, and enhancing immunity. In this technical solution, the biotransformation of plant polysaccharides is effectively improved, promoting the production of new active substances, and reducing toxic side effects.

[0032] Further, in S2, the crude polysaccharide is deproteinized and precipitated with alcohol to obtain a purified mixed polysaccharide; the purified mixed polysaccharide is loaded onto a DEAE-52 ion exchange column for chromatography, and then eluted with sodium chloride solution at a flow rate of 1 mL / min and the eluate is collected, one tube is collected every 10 minutes, and the eluates from tubes 0 to 20 are collected to obtain PPs-1; PPs-1 is loaded onto a G-100 dextran gel column, and then eluted with water at a flow rate of 5 mL / min and the eluate is collected, one tube is collected every 10 minutes, and the eluates from tubes 0 to 15 are collected to obtain PPs-1A; PPs-1 A is loaded onto a G-75 dextran gel column, and then eluted with water at a flow rate of 5 mL / min and the eluate is collected, one tube is collected every 10 minutes, and the eluates from tubes 11 to 20 are collected to obtain PPs-1Aa.

[0033] In summary, the beneficial effects of this technical solution are:

[0034] (1) Provided is a D-galacturonan homogenous pectin polysaccharide with α-glucosidase inhibitory activity, which is expected to replace the conventional drugs in the prior art and overcome the defects of the existing α-glucosidase inhibitors.

[0035] (2) The α-glucosidase inhibition rate of the D-galacturonic acid polysaccharide homogenous pectin polysaccharide in this scheme can reach 80%, IC 50 The value can reach 0.128 mg / mL, which is better than the positive control acarbose, and lays a foundation for further research on drugs for the treatment of diabetes.

[0036] (3) The D-galacturonan homogeneous pectin polysaccharide of this scheme can be produced by microbial fermentation. This method is low-cost, simple to operate, and highly safe. The use of fermentation to extract soluble polysaccharides from pomelo peel can improve the physicochemical properties of the polysaccharide and increase its purity. Generally, natural plant polysaccharides are mostly extracted by hot water extraction, chemical extraction, enzymatic extraction, etc. Microbial fermentation has more advantages than traditional methods.

[0037] (4) This technical solution provides a new comprehensive utilization method for Liangping pomelo natural resources, which improves the medicinal value of Liangping pomelo, can further make full use of pomelo peel resources, and improve the comprehensive utilization efficiency of Liangping pomelo raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a typical appearance image of the crude polysaccharide of Experimental Example 1.

[0039] Figure 2 This is a typical appearance image of the citronella polysaccharide PPs-1Aa of Experimental Example 2.

[0040] Figure 3The results of the α-glucosidase inhibition activity test of Experimental Example 2 are as follows (mean ± standard deviation, the experiment was repeated 3 times, and 6 concentration gradients were selected during the test).

[0041] Figure 4 This is the infrared spectrum detection result of citronella peel polysaccharide PPs-1Aa in Experimental Example 3.

[0042] Figure 5 This is the liquid gel chromatography test result of citronellal polysaccharide PPs-1Aa in Experimental Example 3.

[0043] Figure 6 The results of the monosaccharide composition analysis of citronella peel polysaccharide PPs-1Aa and the mixed standard in Experimental Example 3 are shown.

[0044] Figure 7 The citronellal polysaccharide PPs-1Aa of Experimental Example 3 1 H-NMR(A) and 13 C-NMR(B) spectrum. DETAILED DESCRIPTION

[0045] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial sources.

[0046] Example 1: Extraction of crude polysaccharides

[0047] The extraction method of crude polysaccharides refers to the inventor's previously published paper "Gu Xin, Extraction of Insoluble Dietary Fiber from Liangping Pomelo Peel by Fermentation and Study on Its Physicochemical Properties, Food Science and Technology, 2021, Vol. 46, No. 05". However, in this technical solution, the object of extraction is changed from insoluble dietary fiber to soluble pomelo peel polysaccharide, and the extraction method also changes accordingly. The general process is as follows: Liangping pomelo peel without rot and insect pests is selected for cleaning, and the yellow outer peel is removed by mechanical method. The remaining white inner peel is cut into 1cm pieces. 3 The lumps are then dried in a 55°C oven. After drying and grinding, grapefruit peel powder (80 mesh) is obtained. Accurately weigh 100g of grapefruit peel powder into a conical flask. Based on the conditions of a single-factor experiment or an orthogonal experiment, an appropriate liquid-to-solid ratio of distilled water is added. Then, 2% skim milk powder and 1.5% white sugar are added. After mixing evenly, the mixture is placed in an autoclave for sterilization. After sterilization, the mixture is removed from the autoclave, cooled to room temperature, and then the bacterial strain is added for fermentation. After fermentation is complete, the fermentation product is filtered, and the liquid phase is collected to obtain crude polysaccharide.

[0048] The optimal process parameters for extracting pomelo peel polysaccharides from pomelo peel powder are as follows: a solid-liquid ratio of 20.5 mL / g, an inoculum size of 10%, a fermentation time of 62.5 hours, and a fermentation temperature of 41°C. Under these conditions, the polysaccharide yield was 15.5%. The optimized parameters effectively increased the yield of pomelo peel polysaccharides. The strains used were Lactobacillus bulgaricus and Streptococcus thermophilus, both conventional strains purchased directly from Beijing Chuanxiu International Trading Co., Ltd. The powdered strains were mixed in a 1:1 mass ratio and then added to the fermentation system.

[0049] In previous studies, the inventors used enzymatic hydrolysis to extract citrus peel polysaccharides, see the literature "Gu Xin, Extraction, structural analysis and antioxidant capacity of Liangping pomelo citrus peel polysaccharides, Food and Fermentation Industries, 2021, 47(7): 137-145.". The optimal yield of polysaccharides obtained by this method is about 5%. This technical solution introduces the fermentation method into the extraction process of citrus peel polysaccharides for the first time, increasing the yield of crude citrus peel polysaccharides to 15.5%. Although the microbial fermentation method is time-consuming and takes longer than the enzymatic hydrolysis method, the fermentation method is a pollution-free, mild and more cost-effective method than the enzymatic hydrolysis method, and it can also increase the yield of polysaccharides.

[0050] Example 2: Separation and screening of polysaccharide components

[0051] In this embodiment, the crude polysaccharide obtained by extraction was subjected to simple purification treatment by alcohol precipitation and deproteinization. The citrus polysaccharide was purified by three steps using DEAE-52 cellulose column, G-100 polysaccharide gel column and G-75 polysaccharide gel column in sequence with α-glucosidase inhibition ability as an indicator. Five polysaccharide components (PPs-1, PPs-2, PPs-1A, PPs-1B, PPs-1Aa) were screened. It was proved that the above-mentioned polysaccharide components all had certain α-glucosidase inhibition ability, among which PPs-1Aa had the best effect, and its effect was close to that of acarbose, a conventional drug for treating diabetes. The PPs-1Aa polysaccharide component was obtained as a white flocculent amorphous substance, see Figure 2 The specific operation process is as follows:

[0052] (1) Preliminary purification of citronella polysaccharide

[0053] The crude polysaccharide obtained in Example 1 was deproteinized using the conventional Savag method. Due to the slightly high protein content in pomelo peel, deproteinization was required 3-5 times. The polysaccharide was then extracted with three times the volume of anhydrous ethanol. After standing for 24 hours, the precipitate was collected by centrifugation and freeze-dried to obtain crude pomelo peel polysaccharide (named PPs). The preliminarily purified polysaccharide was obtained. See the following for a photo of its appearance. Figure 1The crude polysaccharide was subjected to alcohol precipitation and deproteinization purification. To test its impurity removal effect, a UV full-wave bed-length automatic detector was used to detect it at 200nm-400nm. No obvious peak was found at 260nm, indicating that its impurity removal effect was good.

[0054] (2) Column purification

[0055] DEAE-52 ion exchange column chromatography

[0056] Weigh 50 mg of crude polysaccharide powder extracted by lactic acid bacteria fermentation, dissolve it in 10 mL of deionized water, centrifuge at 8000 r / min for 10 min, collect the supernatant, filter it through a 0.45 μL water filter membrane, load it onto a chromatography column, then elute with 0.05 mol / L sodium chloride solution, and collect the eluate. The chromatographic column used in this step is a DEAE-52 cellulose column (2.5×60 cm), set the flow rate to 1 mL / min, and collect one tube every 10 min. Draw the elution curve using the phenol-sulfuric acid method, and collect the purified components according to the elution curve. The collected eluate is freeze-dried to obtain polysaccharide powder. The collected purified components (polysaccharide powder) are screened for α-glucosidase inhibition ability, and the polysaccharide components with better ability are selected for the next step.

[0057] Sephadex-G-100 gel column chromatography

[0058] The sample was loaded onto a chromatography column and then eluted with ultrapure water. The chromatographic column was Sephadex-G-100 (2.5×60cm, G-100 dextran gel column), the flow rate was 5ml / min, and one tube was collected every 10 minutes. The elution curve was drawn using the phenol-sulfuric acid method, and the purified components were collected according to the elution curve. The collected eluate was freeze-dried to obtain polysaccharide powder. The collected purified components (polysaccharide powder) were screened for α-glucosidase inhibition ability, and the polysaccharide components with better ability were selected for the next step.

[0059] Sephadex-G-75 gel column chromatography

[0060] The chromatographic column packing was replaced with Sephadex-G-75 (2.5×60 cm, G-75 dextran gel column), and other technical parameters were the same as those of Sephadex-G-100 chromatography. The citronellol polysaccharide powder collected above was treated in the same manner as above.

[0061] After column purification, five polysaccharide components (PPs-1, PPs-2, PPs-1A, PPs-1B, and PPs-1Aa) were collected from PPs. The specific process is as follows: PPs was dissolved in deionized water and then loaded onto a DEAE-cellulose column. The eluates from tubes 0-20 were collected to obtain PPs-1, and the eluates from tubes 75-100 were collected to obtain PPs-2. PPs-1 was loaded onto a G-100 dextran gel column. The eluates from tubes 0-15 were collected to obtain PPs-1A. The eluates from tubes 25-35 were collected to obtain PPs-1B. PPs-1A was loaded onto a G-75 dextran gel column. The eluates from tubes 11-20 were collected to obtain PPs-1Aa.

[0062] (3) α-glucosidase inhibitory activity assay

[0063] α-glucosidase inhibitory activity was determined using a 96-well microplate reader, using PNPG as the detection substrate. 240 μL of a reagent mixture was added to a 96-well microplate, which included 120 μL of 0.5 mol / L phosphate buffer solution (pH = 6.7), 50 μL of substrate solution (0.9 mg / mL PNPG solution dissolved in 0.5 mol / L phosphate buffer), 50 μL of enzyme solution (0.5 μ / ml α-glucosidase solution dissolved in 0.5 mol / L phosphate buffer containing 0.2% BSA), and 20 μL of the sample solution to be tested (the eluate was collected, freeze-dried, and then dissolved according to the specified concentration gradient to calculate the IC 50 The plate was then shaken to mix the solution, sealed, and placed in a 37°C incubator for 1 hour. After completion, the plate was removed and 50 μL of 0.67 mol / L Na₂CO₃ was added to terminate the reaction. This reaction releases a certain amount of PNP, which has a maximum absorption peak at 405 nm. Within a certain concentration range, its absorbance is positively correlated with the α-glucosidase inhibition rate. In the blank control group, the sample volume was replaced with distilled water, and in the background subtraction group, the substrate and enzyme solutions were replaced with 100 μL of phosphate buffer solution.

[0064] The calculation formula of α-glucosidase inhibition rate is:

[0065] AGA(%)=[1-(A1-A2) / A3]×100%

[0066] Wherein, AGA is the α-glucosidase inhibition rate, %; A1 is the absorbance value of the sample group; A2 is the absorbance value of the background group; A3 is the absorbance value of the blank group.

[0067] The inhibition rate of the PPs-1Aa polysaccharide component at a concentration of 0.5 mg / mL can reach 80%, while the inhibition rates of PPs, PPs-1, PPs-2, PPs-1A, and PPs-1B are 56%, 62%, 34%, 66%, and 23%, respectively.

[0068] The IC values ​​of various polysaccharides were detected and calculated. 50 For experimental results, see Figure 3 The α-glucosidase inhibitory effect of PPs-1Aa polysaccharide was the best (IC 50 The α-glucosidase inhibitory activity of PPs without column purification was suboptimal, and the inhibitory effects of PPs-1, PPs-2, PPs-1A, and PPs-1B were also inferior to those of PPs-1Aa. The PPs-1Aa polysaccharide fraction is a potential alternative to conventional α-glucosidase inhibitors and is worthy of further research and promotion.

[0069] Example 3: Structural Identification of Pomelo Peel Polysaccharide PPs-1Aa

[0070] (1) Infrared spectroscopy detection of citronellol polysaccharides

[0071] By scanning the infrared spectrum of the purified sample (PPs-1Aa), it was found that the purified polysaccharide had obvious polysaccharide characteristic glycosidic bonds and some other bonding structure information. -1 The smaller absorption peak is due to the CH stretching vibration in CH2, so polysaccharides will show similar absorption peaks at these two locations, 1741 cm -1 The absorption peak at may be the characteristic peak of acetyl or C=O in carboxylate, indicating that the sample may contain uronic acid. Figure 2 .

[0072] (2) Molecular weight detection of citronellal polysaccharides

[0073] The molecular weight of the polysaccharide was measured by liquid gel chromatography (GPC), and the molecular weight of the purified polysaccharide was 42.8 kDa. The GPC spectrum showed that the sub-fractions showed symmetrical peaks, indicating that the components were homogeneous. Figure 3 .

[0074] (3) Determination of monosaccharide composition of citronellal polysaccharides

[0075] The monosaccharide composition of the sample was determined using 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization and HPLC. The monosaccharide composition of the purified polysaccharide was determined to be primarily composed of galacturonic acid, galactose, rhamnose, glucuronic acid, and glucose. Galacturonic acid accounted for the majority of the sample and had the lowest Rha / Gal value, suggesting that the polysaccharide is a (HG structure) D-galacturonic acid homogeneous pectin polysaccharide. See the graph for the test results of the mixed standard and citrus peel polysaccharide. Figure 4 . According to calculation, the molar percentage of monosaccharides contained in it is: galacturonic acid 86.8%: galactose 3.87%: rhamnose 4.23%: glucuronic acid 1.11%: glucose 0.57%.

[0076] (4) Bond and structure analysis of citronellal polysaccharides

[0077] 1.00 g of the sample was taken for methylation analysis to obtain its corresponding bonding structure. GC-MS results showed that the main chain of the sample polysaccharide was mainly composed of 4-Gal(p)-UA, as shown in Table 1.

[0078] Table 1: Bonding structure analysis results of polysaccharide samples

[0079] Bonding structure derivative Molecular weight Mole percentage t-Gal(p)-UA 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl galactitol 325 8.04 4-Gal(p)-UA 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl galactitol 353 86.09 4-Glc(p) 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl glucitol 351 1.00 6-Gal(p) 1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl galactitol 351 0.88 3,4-Gal(p) 1,3,4,5-tetra-O-acetyl-2,6-di-O-methyl galactitol 379 1.91 4,6-Glc(p)-UA 1,4,5,6-tetra-O-acetyl-2,3-di-O-methyl glucitol 381 2.07

[0080] (5) Nuclear magnetic resonance analysis

[0081] 1 H NMR spectra can be used to determine the glycosidic bond configuration in the polysaccharide structure. Usually, the proton signal of the anomeric carbon of α-type glycoside is greater than δ5.0, while the proton signal of the anomeric carbon of β-type glycoside is less than δ5.0. Figure 7 As shown, the polysaccharide belongs to the anomeric carbon of β-glycoside. According to NMR analysis and the results obtained in the above steps, it is believed that the polysaccharide is a (HG structure) D-galacturonan homopectin polysaccharide.

[0082] In summary, after impurity removal, the citronellal polysaccharide obtained by lactic acid bacteria fermentation was deduced to be an acidic polysaccharide with pyranose rings connected by β-glycosidic bonds, as determined by infrared spectroscopy. With α-glucosidase inhibition as the screening target, a citronellal polysaccharide with excellent α-glucosidase inhibition was obtained through multi-step purification and screening. GPC results showed a uniform and symmetrical single peak, demonstrating the high purity of the polysaccharide. Monosaccharide composition analysis, molecular weight determination, and NMR analysis of the purified citronellal polysaccharide were performed, indicating that the polysaccharide is a (HG structure) D-galacturonan homogeneous pectin polysaccharide. Based on the comprehensive analysis of the above experimental results, it can be inferred that the polysaccharide has a skeleton structure composed of →4)-α-GalpA-(1→) and →4)-α-GalpA-6-O-methyl-(1→) (as shown in formula (1)).

[0083]

[0084] The above is only an embodiment of the present invention, and the common knowledge such as the specific technical solutions and / or characteristics in the solution are not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the description can be used to interpret the content of the claims.

Claims

1. A D-galacturonan homogeneous pectin polysaccharide, characterized in that: Its monosaccharide composition mainly includes galacturonic acid, galactose, rhamnose, glucuronic acid and glucose; its skeleton structure is composed of →4)-α-GalpA-(1→ and →4)-α-GalpA-6-O-methyl-(1→; It is prepared by the following method: S1 Preparation of crude polysaccharide: The white inner peel of Liangping pomelo peel is taken and crushed to obtain pomelo peel powder; water, a carbon source, and a bacterial strain are then added to obtain crude polysaccharide through fermentation; the bacterial strain is composed of Lactobacillus bulgaricus and Streptococcus thermophilus; S2 purification: After the crude polysaccharide is subjected to alcohol precipitation and deproteinization treatment, a purified mixed polysaccharide is obtained; the purified mixed polysaccharide is loaded onto a DEAE-52 ion exchange column for chromatography, and then eluted with sodium chloride solution at a flow rate of 1 mL / min and the eluate is collected, one tube is collected every 10 minutes, and the eluates from tubes 0 to 20 are collected to obtain PPs-1; PPs-1 is loaded onto a G-100 dextran gel column, and then eluted with water at a flow rate of 5 mL / min and the eluate is collected, one tube is collected every 10 minutes, and the eluates from tubes 0 to 15 are collected to obtain PPs-1A; PPs-1A is loaded onto a G-75 dextran gel column, and then eluted with water at a flow rate of 5 mL / min and the eluate is collected, one tube is collected every 10 minutes, and the eluates from tubes 11 to 20 are collected to obtain PPs-1Aa.

2. A D-galacturonan homogeneous pectin polysaccharide according to claim 1, characterized in that: The molar ratios of galacturonic acid, galactose, rhamnose, glucuronic acid, and glucose are 86.8:3.87:4.23:1.11:0.

57.

3. A D-galacturonan homogeneous pectin polysaccharide according to claim 2, characterized in that: The residues and molar percentages obtained from the methylation analysis are: 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl galactitol 8.04%, 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl galactitol 86.09%, 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl glucitol 1.00%, 1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl galactitol 0.88%, 1,3,4,5-tetra-O-acetyl-2,6-di-O-methyl galactitol 1.91%, 1,4,5,6-tetra-O-acetyl-2,3-di-O-methyl glucitol 2.07%.

4. The D-galacturonan homogeneous pectin polysaccharide according to claim 3, characterized in that: The bonding structure of 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl galactitol is t-Gal(p)-UA; The bonding structure of 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl galactitol is 4-Gal(p)-UA; The bonding structure of 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl glucitol is 4-Glc(p); The bonding structure of 1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl galactitol is 6-Gal(p); The bonding structure of 1,3,4,5-tetra-O-acetyl-2,6-di-O-methyl galactitol is 3,4-Gal(p); The bonding structure of 1,4,5,6-tetra-O-acetyl-2,3-di-O-methyl glucitol is 4,6-Glc(p)-UA.

5. A D-galacturonan homogeneous pectin polysaccharide according to claim 4, characterized in that: Its molecular weight is 42.8 kDa.

6. The D-galacturonan homogeneous pectin polysaccharide according to claim 5, characterized in that: IC of its inhibitory effect on α-glucosidase 50 The value is 0.128 mg / mL.

7. A method for preparing D-galacturonan homogeneous pectin polysaccharide, characterized in that: The method includes the following steps: S1 Preparation of crude polysaccharide: The white inner peel of Liangping pomelo peel is taken and crushed to obtain pomelo peel powder; water, a carbon source, and a bacterial strain are then added to obtain crude polysaccharide through fermentation; the bacterial strain is composed of Lactobacillus bulgaricus and Streptococcus thermophilus; S2 purification: After the crude polysaccharide is subjected to alcohol precipitation and deproteinization treatment, a purified mixed polysaccharide is obtained; the purified mixed polysaccharide is loaded onto a DEAE-52 ion exchange column for chromatography, and then eluted with sodium chloride solution at a flow rate of 1 mL / min and the eluate is collected, one tube is collected every 10 minutes, and the eluates from tubes 0 to 20 are collected to obtain PPs-1; PPs-1 is loaded onto a G-100 dextran gel column, and then eluted with water at a flow rate of 5 mL / min and the eluate is collected, one tube is collected every 10 minutes, and the eluates from tubes 0 to 15 are collected to obtain PPs-1A; PPs-1A is loaded onto a G-75 dextran gel column, and then eluted with water at a flow rate of 5 mL / min and the eluate is collected, one tube is collected every 10 minutes, and the eluates from tubes 11 to 20 are collected to obtain PPs-1Aa.

8. The method for preparing D-galacturonan homogeneous pectin polysaccharide according to claim 7, characterized in that: In S1, the fermentation conditions were as follows: solid-liquid ratio 20.5 mL / g, inoculation size 10% of the mass of pomelo peel powder, fermentation time 62.5 h, and fermentation temperature 41°C; the carbon sources included skim milk powder 2% of the mass of pomelo peel powder and white sugar 1.5% of the mass of pomelo peel powder.

9. Use of a D-galacturonan homopectin polysaccharide according to any one of claims 1 to 6 in the preparation of a medicament for treating diabetes.

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