Portulaca oleracea polysaccharide as well as preparation method and application thereof in livestock breeding

Abstract

The invention discloses a preparation method of portulaca oleracea polysaccharide. The preparation method comprises the following steps: S1, pretreating portulaca oleracea; s2, degreasing treatment; s3, extraction of crude purslane polysaccharide; s4, removal of alcohol soluble impurities; s5, removing pigments; s6, removal of protein; s7, removing small molecular impurities; and S8, separation and purification. The purslane polysaccharide extracted by the method disclosed by the invention is applied to preparation of a feed additive which has a growth promoting effect and / or a diarrhea preventing and treating effect on piglets. The extraction method provided by the invention can effectively improve the extraction efficiency of the active components, and ensures that the extract has relatively strong biological activity and protective effect. The portulaca oleracea polysaccharide disclosed by the invention has relatively strong water retention effect and thermal stability, good solubility and good adsorption effect, has a remarkable growth promoting effect on piglets, can be used for remarkably reducing the diarrhea rate of weaned piglets, and has a good application prospect in livestock breeding.

Description

Technical Field

[0001] The invention relates to purslane polysaccharide, a preparation method thereof and application thereof in animal husbandry, and belongs to the technical field of animal husbandry. Background Art

[0002] Portulaca oleracea L. is an annual herbaceous plant of the genus Portulacaceae. It is drought- and flood-tolerant, resilient, and rich in nutritional value, and is widely distributed throughout my country. The entire plant is used medicinally. Modern pharmacology suggests that Portulaca oleracea contains a variety of effective bioactive components, including alkaloids, polysaccharides, flavonoids, and fatty acids, with polysaccharides typically accounting for 5% to 15% of its dry weight. Portulaca oleracea L. polysaccharides (POPs), as natural polysaccharides, exhibit antioxidant, immune-modulating, and intestinal health-enhancing properties. In recent decades, POPs have garnered considerable attention due to their non-toxicity, high abundance, and numerous known beneficial pharmacological effects. The bioactivities and health benefits of POPs have been extensively studied both in vitro and in vivo. The diverse physicochemical properties and structures of POPs, obtained through different extraction and purification methods, contribute to their diverse bioactivities. Summary of the Invention

[0003] In view of the above-mentioned prior art, the present invention provides a purslane polysaccharide, a preparation method thereof, and application thereof in animal husbandry.

[0004] The present invention is achieved through the following technical solutions:

[0005] A method for preparing purslane polysaccharide comprises the following steps:

[0006] S1. Pretreatment of purslane: taking dried purslane, crushing it to obtain purslane powder, and setting aside;

[0007] S2. Degreasing treatment: adding the purslane powder to petroleum ether and subjecting to Soxhlet reflux extraction for 3 to 4 hours; after the reflux extraction, taking out the sample, washing it with water, and drying it to obtain defatted purslane powder;

[0008] S3. Extraction of crude purslane polysaccharide: Mix defatted purslane powder with water, and boil for 1.5 to 2 hours (water may be added during the process); then perform ultrasonic-assisted extraction; after the ultrasonic-assisted extraction is completed, stir for 15 to 20 minutes; and filter. The filtrate is the crude purslane polysaccharide solution;

[0009] S4. Removal of alcohol-soluble impurities: The crude purslane polysaccharide solution is concentrated, anhydrous ethanol or ethanol solution is added to make the ethanol concentration 75% to 85% (volume percentage), and the solution is allowed to stand for alcohol precipitation; centrifugation is performed, and the precipitate is collected;

[0010] S5, pigment removal: dissolve the precipitate in step S4 in water, add AB-8000 macroporous resin, stir at 35-50°C for 4-5 hours for decolorization; filter to obtain a decolorized solution;

[0011] S6. Protein removal: Add Sevag reagent to the decolorizing solution and stir for 30 to 50 minutes for deproteinization; let stand to separate the layers, and the lower layer is the deproteinized solution; repeat the deproteinization process 6 to 8 times; the Sevag reagent is composed of chloroform and n-butanol in a volume ratio of 5:1;

[0012] S7. Removal of small molecule impurities: dialyze the deproteinized solution in an 8000-14000 KDa dialysis bag; after dialysis, freeze-dry to obtain crude purslane polysaccharide;

[0013] S8. Isolation and purification of purslane polysaccharide: dissolve the crude purslane polysaccharide in water, load it onto a DEAE-52 cellulose anion exchange column and elute it with a NaCl solution, collect the eluate of 0.4 M NaCl solution; dialyze the eluate in an 8000-14000 KDa dialysis bag; after dialysis, freeze-dry to obtain refined purslane polysaccharide.

[0014] Furthermore, in step S1, the dried purslane is obtained by the following method: taking fresh whole purslane, washing and drying it, and drying it in an oven at a low temperature of 50-60° C. to a constant weight.

[0015] Furthermore, in step S1, the powder is crushed and then passed through a 200-mesh sieve.

[0016] Furthermore, in step S2, the material-liquid ratio is 1:20 (g:ml).

[0017] Furthermore, in step S3, the material-liquid ratio of defatted purslane powder to water is 1:30 (g:ml).

[0018] Furthermore, in step S3, the parameters of ultrasound-assisted extraction are: temperature 55° C. to 60° C., power 400 W, and time 40 to 50 minutes.

[0019] Furthermore, in step S3, the extraction (boiling extraction, ultrasonic-assisted extraction, stirring) is repeated three times, and the filtrates from the three times are combined to obtain a crude purslane polysaccharide solution.

[0020] Furthermore, in step S4, the solution is concentrated by stirring and heating at a uniform speed to volatilize the water, and concentrating the solution to 1 / 10 to 1 / 8 of the original volume.

[0021] Furthermore, in step S4, the specific method of standing and precipitating with alcohol is: standing at 4° C. for 8 to 12 hours.

[0022] Furthermore, in step S4, the precipitate is treated as follows: the precipitate is washed with 90% ethanol solution (volume percentage), anhydrous ethanol, and acetone in sequence, each washing 1 to 3 times, and then dried.

[0023] Furthermore, in step S5, the mass ratio of crude purslane polysaccharide to water is 1:200, and the amount of AB-8000 macroporous resin added is 60 g / L.

[0024] Furthermore, in step S6, the volume ratio of the decolorizing solution to the Sevag reagent is 5:1.

[0025] The purslane polysaccharide extracted by the above method is used in animal husbandry as / in the preparation of a feed additive that has a growth-promoting effect and / or a diarrhea-preventing effect on piglets.

[0026] Furthermore, in specific applications, purslane polysaccharide is added to the basic feed of piglets at a ratio of 0.05% to 0.35% (weight percentage), preferably 0.1% or 0.3%, and fed to piglets under normal conditions, which can achieve good diarrhea prevention and treatment effects and growth promotion effects.

[0027] The purslane polysaccharide extracted by the above method has the following characteristics:

[0028] (1) The efficiency of crude purslane polysaccharide extracted from defatted purslane powder is about 21% (W / W); the purity of refined polysaccharide reaches 75.4%;

[0029] (2) The purslane polysaccharide extracted by the method of the present invention is mainly a water-soluble polysaccharide in the whole plant of purslane;

[0030] (3) The purslane polysaccharide extracted by the method of the present invention is mainly composed of acidic polysaccharides;

[0031] (4) The purslane polysaccharide extracted by the method of the present invention is a mixed polysaccharide composed of two components with relative molecular weights of 3.35 KDa and 265.91 KDa, accounting for 76.9% and 23.1% respectively, with the component with a molecular weight of 3.35 KDa being the main component;

[0032] (5) The purslane polysaccharide extracted by the method of the present invention is mainly composed of arabinose, mannose and xylose; it is a cell wall polysaccharide;

[0033] (6) The purslane polysaccharide extracted by the method of the present invention has a high content of uronic acid groups;

[0034] (7) The weight of water bound to the purslane polysaccharide extracted by the method of the present invention accounts for 29.5% of the original weight of the polysaccharide, and has strong water retention and thermal stability;

[0035] (8) The purslane polysaccharide extracted by this method has a smooth and dense surface, a compact flaky structure, and good stability; at the same time, it shows different degrees of curling and looks softer, indicating that the purslane polysaccharide has good solubility.

[0036] (9) The purslane polysaccharide extracted by this method exhibited a sponge-like appearance structure after being dissolved in water, indicating that the polysaccharide may have good adsorption effect.

[0037] (10) The purslane polysaccharide extracted by this method can be used as a feed additive and added to a variety of feeds for a variety of animals at appropriate concentrations to prevent animal diarrhea and promote animal growth, or to improve animal immunity and reduce stress responses.

[0038] The present invention's purslane polysaccharide extraction method combines hot water extraction with ultrasound-assisted extraction, effectively improving the extraction efficiency of the active ingredient and ensuring that the extract has strong biological activity and protective effects. The present invention's purslane polysaccharide has broad application prospects in animal husbandry, and can prevent diarrhea and promote animal growth, or enhance animal immunity and reduce stress responses.

[0039] Various terms and phrases used herein have the general meanings that are well known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 : Elution curve of purslane polysaccharide.

[0041] Figure 2 : Photographs of freeze-dried purslane polysaccharide.

[0042] Figure 3 : Scanning image of purslane polysaccharide at 200~500nm.

[0043] Figure 4 : Schematic diagram of the molecular weight detection results of Portulaca oleracea polysaccharide.

[0044] Figure 5 : Schematic diagram of the monosaccharide composition analysis and identification results of Portulaca oleracea polysaccharide.

[0045] Figure 6 : Schematic diagram of thermogravimetric analysis results of Portulaca oleracea polysaccharide.

[0046] Figure 7 : Schematic diagram of Fourier transform infrared spectroscopy identification results of Portulaca oleracea polysaccharide.

[0047] Figure 8 : SEM images of freeze-dried powder of purslane polysaccharide, where Ⅰ.500×; Ⅱ.1000×; Ⅲ.10000×.

[0048] Figure 9 : SEM examination images of the aqueous solution of purslane polysaccharide, where Ⅰ.3000×; Ⅱ.5000×; Ⅲ.200000×.

[0049] Note: Figure 5 、 Figure 8 、 Figure 9 The unclearness of some lines and texts is caused by software generation, but does not affect the content expressed in the drawings and does not affect the understanding of the present invention by those skilled in the art. DETAILED DESCRIPTION

[0050] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will appreciate that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0051] Unless otherwise specified, the instruments, reagents, and materials used in the following examples are all conventional instruments, reagents, and materials available in the prior art and can be obtained through regular commercial channels. The experimental methods and detection methods used in the following examples are all conventional experimental methods and detection methods available in the prior art, unless otherwise specified.

[0052] Example 1 Isolation, purification and structural characterization of purslane polysaccharide

[0053] 1. Materials and Equipment

[0054] 1.1 Experimental Materials

[0055] This experiment selected freshly bloomed purslane from a plantation in Beijing, China. After harvesting, the purslane was cleaned and dried.

[0056] 1.2 Experimental Reagents

[0057] The main reagents used in the experiment are shown in Table 1.

[0058] Table 1 Main reagents used in the experiment

[0059]

[0060]

[0061] 1.3 Experimental instruments and equipment

[0062] The main instruments and equipment involved in the experiment are: ultra-high-speed crusher, ultrasonic cleaner, centrifuge, freeze dryer, scanning electron microscope (SEM), thermogravimetric analyzer (TGA), gas chromatography-mass spectrometer (GC-MS), gel permeation chromatography system (GPC), and Fourier transform infrared spectrometer (FT-IR).

[0063] 2. Experimental Methods

[0064] 2.1 Extraction of Purslane Crude Polysaccharide

[0065] Ultrasound-assisted hot water extraction was used to extract polysaccharides from the whole plant of Portulaca oleracea. The process was as follows:

[0066] Wash the whole plant of fresh Purslane with clean water, control the moisture, and dry it in a 50°C oven to constant weight. Grind it into powder using an ultra-high-speed grinder and pass it through a 200-mesh sieve.

[0067] The sieved powder sample was placed in petroleum ether with a boiling range of 30 to 60° C. and refluxed for 3 hours to remove lipids. After reflux extraction, the sample was taken out, washed with water, and dried at a low temperature of 50° C. to remove residual solvent, thereby obtaining defatted purslane powder.

[0068] The defatted purslane powder was mixed with distilled water at a solid-liquid ratio of 1:30, and the mixture was stirred and boiled for 2 hours at a uniform speed, with water added in the middle; ultrasonic-assisted extraction was then performed (power 400W, temperature 60°C, time 45 minutes); after the ultrasonic-assisted extraction was completed, the mixture was stirred for 20 minutes, filtered, and the filtrate was collected; the extraction (boiling extraction, ultrasonic-assisted extraction, stirring) was repeated 3 times; the filtrates from the 3 times were collected and combined to obtain a purslane crude polysaccharide solution.

[0069] The crude purslane polysaccharide solution was stirred and heated at a uniform speed and concentrated to 1 / 10 of the original volume, anhydrous ethanol was added to a final concentration of 80%, and the solution was allowed to stand at 4°C for 10 hours; the solution was centrifuged (4500 rpm, 15 minutes), the precipitate was collected, and washed with 90% ethanol solution (volume percentage), anhydrous ethanol, and acetone in sequence, each washed twice, and air-dried in a ventilated place to obtain powdered crude purslane polysaccharide.

[0070] The crude purslane polysaccharide is dissolved in water (the mass ratio of the crude purslane polysaccharide to water is 1:200), AB-8 macroporous resin is added (to a concentration of 60 g / L), and the mixture is stirred at 50° C. for 5 hours for decolorization; the mixture is filtered to obtain a decolorized solution; Sevag reagent is added to the decolorized solution (the volume ratio of the decolorized solution to the Sevag reagent is 5:1), and the mixture is stirred for 40 minutes for deproteinization; the mixture is allowed to stand for stratification, the upper layer of Sevag reagent is removed, and the lower layer solution is the deproteinized solution; Sevag reagent is added again to the deproteinized solution for deproteinization, and the process is repeated 8 times; the Sevag reagent is composed of chloroform and n-butanol in a volume ratio of 5:1; the deproteinized solution is dialyzed in an 8000-14000 KDa dialysis bag for 48 hours (the dialysis water solution is replaced several times during the period), and then freeze-dried at -80° C. to obtain a white freeze-dried powder of the crude purslane polysaccharide.

[0071] 2.2 Isolation and purification of purslane polysaccharides

[0072] In order to obtain higher purity of purslane polysaccharide, DEAE-52 cellulose anion chromatography column was used to further purify the polysaccharide freeze-dried powder. First, the freeze-dried polysaccharide powder was dissolved in warm water to prepare a 1 mg / ml polysaccharide loading solution, which was loaded onto the DEAE-52 cellulose anion exchange column. Distilled water, 0.1 M, 0.2 M, 0.4 M and 0.6 M NaCl solutions were used for elution in sequence. The elution curve is shown in Figure 2. Figure 1 As shown. 0.4M NaCl solution was selected as the eluent to collect all the polysaccharide components in purslane. The eluate was dialyzed for 48 hours using a dialysis bag with a cutoff of 8000-14000KDa to remove salt ions, and then freeze-dried at -80℃ to obtain refined purslane polysaccharides, as shown. Figure 2 shown.

[0073] 2.3 Determination of chemical composition of purslane polysaccharides

[0074] The total polysaccharide content of Portulaca oleracea polysaccharides was determined using the phenol-sulfuric acid method. An appropriate amount of polysaccharide powder was weighed to prepare a 1 mg / ml solution. Using a pipette, 400 μL of sample solution was added, 200 μL of 6% phenol solution was added, and after shaking, 1 mL of concentrated sulfuric acid solution was slowly added. The sample was placed in a 90°C water bath for 10 minutes. The absorbance was measured at 490 nm. A standard curve was prepared using glucose as the standard and the total sugar content was calculated. Scanning detection was performed within the absorbance range of 200–500 nm.

[0075] 2.4 Molecular weight determination of purslane polysaccharide

[0076] The molecular weight distribution of purslane polysaccharide was analyzed by normal temperature / high temperature gel chromatography (GPC) combined with a differential refractive index detector (RID-20). An appropriate amount of purslane polysaccharide sample was dissolved in a suitable solvent to prepare a 1 mg / mL solution. The sample solution was passed through a TSKgel GMPWXL aqueous gel chromatography column (TOSOH) at a flow rate of 1 mL / min, using 0.1N NaN3 and 0.06% NaN3 solution as the mobile phase, the column temperature was set to 35°C, and the mobile phase flow rate was 0.6 mL / min. A differential refractive index detector (RID-20) was used for detection, and SHODEX pullulan polysaccharide standards were used for calibration to calculate the molecular weight distribution of the sample.

[0077] 2.5 Analysis of monosaccharide composition of purslane polysaccharide

[0078] The monosaccharide composition of purslane polysaccharides was analyzed using gas chromatography-mass spectrometry (GC-MS). 50 mg of purslane polysaccharide was hydrolyzed at 100°C for 6 hours with 3 mL of trifluoroacetic acid. 1 mL of the hydrolyzate was dried under nitrogen, and then 2 mL of pyridine and 100 μL of N,O-bis(trimethylsilyl)trifluoroacetamide were added. The mixture was heated to 80°C for 2 hours. After completion of the reaction, the sample was immediately filtered (using a 0.22 μm filter membrane) and used for GC-MS analysis. An Agilent 8860 GC-MS analyzer equipped with an Agilent J&W HP-5ms column (30 m × 0.25 mm, 0.25 μm film thickness) was used for monosaccharide composition analysis. The injection volume was 0.2 μL, the split ratio was 20:1, helium was used as the carrier gas, and the column temperature was programmed from 40°C to 280°C.

[0079] 2.6 Analysis of thermal stability of purslane polysaccharide

[0080] The thermal stability of the samples was determined using a thermogravimetric analyzer (Mettler Toledo TGA2). Approximately 20 mg of the sample was placed in a crucible and then heated from room temperature to 800°C under a nitrogen atmosphere at a flow rate of 50 mL / min and a flow rate of 10°C / min.

[0081] 2.7 Infrared spectroscopy analysis of purslane polysaccharides

[0082] The infrared spectrometer (FT-IR) was used to detect the purslane polysaccharide. An appropriate amount of purslane polysaccharide sample was mixed with dry KBr, ground and pressed into tablets, and the sample was detected by infrared spectrometer at 4000-400 cm -1 The absorption value within the frequency range.

[0083] 2.8 Scanning electron microscopy (SEM) analysis of purslane polysaccharides

[0084] The surface morphology of purslane polysaccharide was observed using scanning electron microscopy. Purslane polysaccharide powder / solution samples were adhered to mica / silicon wafers using a conductive adhesive and then gold powder-sprayed. The surface structure of the powder samples was observed at 500×, 1000×, and 10,000× magnifications at accelerating voltages of 5 kV and 10 kV, respectively. The structure of the polysaccharide solution was observed at 3000×, 5000×, and 20,000× magnifications for the solution samples.

[0085] 2.9 Data Analysis

[0086] All experiments in this study were repeated three times or more. SPSS (Statistical Package for the Social Sciences) was used to perform one-way analysis of variance (ANOVA) and independent sample T-test to analyze the statistical significance of the obtained data, and Graphpad Prism 10 and Origin were used for graphics.

[0087] 3. Results and Analysis

[0088] 3.1 Extraction and purification of purslane polysaccharides

[0089] Polysaccharides were extracted from purslane by combining multiple procedures such as hot water extraction, ethanol precipitation, decolorization and protein removal. The crude polysaccharide yield extracted by this extraction method can reach about 21% (W / W). The polysaccharide components were then separated and purified using anion exchange chromatography using DEAE-Cellulose-52. According to the elution curve, three polysaccharide fractions with different charge densities were eluted by gradually increasing the concentration of sodium chloride (0.1M, 0.2M, 0.4M and 0.6M). It is worth noting that the polysaccharides extracted by this method do not contain neutral polysaccharide components and are mainly composed of acidic polysaccharides. All polysaccharide components were collected with 0.4M NaCl solution, dialyzed and freeze-dried to obtain purslane purified water-soluble polysaccharides (POP). The total sugar content of POP was determined to be 75.4% by the phenol-sulfuric acid method. Purslane polysaccharides are concentrated at 200-500nm. -1 The scanned image is as follows Figure 3 As shown, it can be seen that at 260-280nm -1 No significant absorption peak was detected in the absorbance range of , indicating that the polysaccharide sample did not contain nucleic acid or protein components.

[0090] 3.2 Thermal stability analysis of POP

[0091] Thermogravimetric analysis (TG) results are as follows Figure 6 As shown, two typical weight loss steps are observed in the TG curves of POP when the heating temperature is increased from 30°C to 800°C. Within the temperature range of 50°C to 200°C, POP loses approximately 29.5% of its mass, primarily due to the evaporation of bound water. The relatively large mass loss of POP during this step indicates that the polysaccharide extract has a strong water-retention capacity. In the range of 200°C to 350°C, significant mass loss occurs in the purslane polysaccharide, primarily due to chemical changes such as thermal decomposition and decarboxylation of acid groups.

[0092] 3.3 POP molecular weight distribution

[0093] The molecular weight of purslane polysaccharide was determined by gel permeation chromatography. Figure 4As shown in the figure, the purslane polysaccharide is composed of two components with different molecular weights, accounting for 76.9% and 23.1% respectively; their relative molecular masses (Mw) are 3.35kDa and 265.91kDa, respectively, with an average relative molecular weight of 63.91kDa; their number average molecular weights (Mn) are 0.94kDa and 86.80kDa, respectively, with an average number weight molecular weight of 1.22kDa; and their polydispersity indices (Mw / Mn) are 3.56 and 3.06, respectively, indicating that the purslane polysaccharide is composed of two high molecular weight components and has a certain degree of complexity.

[0094] 3.4 Analysis of Monosaccharide Composition of Portulaca Oleracea Polysaccharides

[0095] The monosaccharide composition was analyzed by gas chromatography-mass spectrometry. Figure 5 As shown. Purslane polysaccharide is composed of 7 different monosaccharides including xylose (Xyl), arabinose (Ara), mannose (Man), galactose (Gal), ribose (Rib), altrose (Alt) and lyxose (Lyx), with the molar ratio of each monosaccharide being 9.24:36.81:20.46:8.46:8.36:8.41:8.26, among which arabinose, mannose and xylose are the main monosaccharide components of the polysaccharide. The detection peak of Ara appears at four different time points, which may be related to the structural characteristics of Ara, such as furanose, pyranose, configuration, etc. Since arabinose and xylose are key monosaccharides of plant cell wall polysaccharides, it is proved that the purslane polysaccharide extracted by this method may be a cell wall polysaccharide.

[0096] 3.5 Infrared spectroscopy analysis of purslane polysaccharides

[0097] The results of Fourier infrared spectroscopy identification of purslane polysaccharide are as follows Figure 7 Fourier transform infrared spectroscopy analysis showed that purslane polysaccharide has typical polysaccharide characteristic absorption peaks, including 3400.6cm -1 (-OH stretching vibration), 2926.7cm -1 (CH stretching and bending vibration), 1615cm -1 (ester carbonyl -COOR or carboxylated ion group COO-, indicating the presence of uronic acid), 1416.8 cm -1 (stretching and bending vibration of CH or OH) and 1028cm -1 (C-O stretching vibration on the guanosyl ring).

[0098] 3.6 Scanning electron microscopy analysis of purslane polysaccharide powder samples

[0099] The surface structure of purslane polysaccharide was analyzed by scanning electron microscopy. Figure 8As shown in the images, at 500×, 1000×, and 10,000× magnifications, the purslane polysaccharide exhibits a smooth, dense, and compact flaky appearance, indicating strong intermolecular interactions and a possible highly branched structure. It is also observed to have varying degrees of curling, appearing softer, suggesting good solubility.

[0100] 3.7 Scanning electron microscopy analysis of purslane polysaccharide aqueous solution

[0101] The structure of the aqueous solution of purslane polysaccharide was analyzed by scanning electron microscopy. Figure 9 As shown, at 3000×, 5000× and 200000× magnifications, purslane polysaccharide exhibited a sponge-like conformation after being dissolved in water, which indicated that purslane polysaccharide had a high adsorption effect after being dissolved in water.

[0102] 4. Summary

[0103] In this study, purslane polysaccharides were extracted and structurally characterized using a combination of water extraction and alcohol precipitation, ultrasound-assisted extraction, color and protein removal, and DEAE cellulose cleanup. The results demonstrated that high-purity purslane polysaccharides were successfully obtained through hot water extraction combined with ethanol precipitation, decolorization, and protein removal, achieving an extraction yield of 21%. Analysis of the total sugar, protein, and nucleic acid contents revealed that the purslane polysaccharide was a pure polysaccharide, devoid of nucleic acids and proteins. The purslane polysaccharide consisted of components with relative molecular masses (Mw) of 3.35 kDa and 265.91 kDa, respectively, indicating that POP is a high-molecular-weight polysaccharide mixture. GC-MS analysis revealed that the purslane polysaccharide was primarily composed of monosaccharides such as arabinose, mannose, xylose, and galactose, consistent with plant cell wall polysaccharides. FT-IR analysis confirmed the functional groups and bonds, suggesting that POP may be an acidic polysaccharide. Thermogravimetric analysis revealed good thermal stability. SEM images show that purslane polysaccharide has a compact lamellar structure and may have a high degree of branching. Overall, purslane polysaccharide has high purity and a complex structure, and has a wide range of application potential.

[0104] Experiment 1: Growth-promoting effect of purslane polysaccharide on piglets and its preventive effect on diarrhea in piglets

[0105] As a natural active ingredient, purslane polysaccharide has shown many application potentials in animal husbandry: (1) It can replace antibiotics and prevent and control diseases by inhibiting pathogenic microorganisms, exerting anti-inflammatory and immunomodulatory effects. POP has a significant inhibitory effect on common pathogenic bacteria such as Escherichia coli and Staphylococcus aureus. (2) It can improve production performance and feed conversion rate by promoting growth performance and regulating metabolic hormones. (3) It can maintain intestinal health and flora balance by inhibiting harmful bacteria, promoting probiotics, and alleviating intestinal inflammation. (4) It can scavenge free radicals, delay cell damage and protect organ function through antioxidant and anti-stress effects. Therefore, the present invention studies its growth-promoting effect on piglets and its preventive effect on piglet diarrhea, as shown below.

[0106] 1. Materials and Equipment

[0107] 1.1 Experimental Materials

[0108] In this experiment, the purslane polysaccharide prepared according to the method of Example 1 was used as the material to test its growth-promoting effect and diarrhea prevention effect.

[0109] 1.2 Experimental Materials

[0110] The main materials used in the experiment are shown in Table 2.

[0111] Table 2 Main materials used in the experiment

[0112] Material name illustrate Portulaca oleracea polysaccharide extract self made Piglet complete nutrition stage compound feed Purchased from Da Bei Nong Company Living water Use after boiling PE gloves conventional latex gloves conventional Face mask conventional

[0113] 1.3 Experimental instruments and equipment

[0114] The main instruments and equipment involved in the experiment are: ultra-high-speed crusher, feed mixer, electronic scale, and electronic balance.

[0115] 1.4 Experimental Animals

[0116] Fifteen healthy weaned local piglets of similar weight were randomly divided into three groups, with five replicates in each group. The three groups of piglets were fed a basal diet (control group), a basal diet plus 0.1% purslane polysaccharide (experimental group A), and a basal diet plus 0.3% purslane polysaccharide (experimental group B), respectively.

[0117] 2. Experimental Methods

[0118] 2.1 Experimental Animal Husbandry and Management

[0119] The experiment was conducted at a breeding pig farm in Fuxing Village, Jinchuan County, Aba Prefecture, Sichuan Province, for 25 days. Pigs were reared and managed according to routine farm procedures. The basal diet used was a locally available Da Beinong piglet compound feed, mixed with hot water, and fed three times daily.

[0120] 2.2 Measurement parameters

[0121] Observe and record the health of the piglets daily, focusing on mortality, culling, and diarrhea. For severe diarrhea, analyze the appearance and color of the feces and, if necessary, collect feces for microscopic examination. Weigh each piglet on an empty stomach on the morning of the first and 25th day after the start of the experiment. Record the feed intake of each piglet. Calculate the average daily weight gain, average daily feed intake, feed-to-weight ratio, mortality rate, and diarrhea rate for each group.

[0122] 2.3 Statistical analysis

[0123] The data were analyzed by variance and multiple comparison using SPSS 17.0 software. P < 0.05 was considered significant difference. The results were expressed as mean ± standard error.

[0124] 3. Results and Analysis

[0125] 3.1 Effects of purslane polysaccharides on growth performance of weaned piglets

[0126] The effect of purslane polysaccharide on the growth performance of weaned piglets is shown in Table 3. As shown in Table 3, when 0.3% purslane polysaccharide was added to the diet, the final weight and average daily gain of the piglets were significantly different from those of the control group (P < 0.05); adding 0.1% purslane polysaccharide also had a more obvious increasing trend on the daily gain of weaned piglets, and the difference between the two addition levels was not significant. It can be seen that adding a certain proportion of purslane polysaccharide to the diet can increase the average daily feed intake of weaned piglets and reduce the feed-to-gain ratio, but there was no significant difference (P > 0.05). Among the two addition concentrations, adding 0.3% had a better effect.

[0127] Table 3 Effects of purslane polysaccharide on growth performance of weaned piglets

[0128] project control group Experimental group A Experimental group B Initial weight / kg 5.14+6.25 4.79+5.61 5.52+6.85 Final weight / kg <![CDATA[15.56+22.80 a ]]> <![CDATA[15.98+23.44 a ]]> <![CDATA[16.61+22.49 a ]]> Average daily weight gain / g <![CDATA[416.80+12.35 a ]]> <![CDATA[262.56+12.68 a ]]> <![CDATA[262.56+11.93 a <!-- 8 -->]]> Average daily feed intake / g <![CDATA[462.60+19.54 a ]]> <![CDATA[477.09+18.50 a ]]> <![CDATA[493.76+19.48 a ]]> Material-to-weight ratio 1.28+0.02 1.97+0.04 1.59+0.07

[0129] Note: Data in the same industry with different lowercase letters in the shoulder indicate significant differences, different uppercase letters in the shoulder indicate extremely significant differences, and no shoulder or the same shoulder indicate insignificant differences.

[0130] 3.2 Effect of purslane polysaccharide on diarrhea in weaned piglets

[0131] The effects of supplementing piglet diets with purslane polysaccharide on the incidence of diarrhea are shown in Table 4. As shown in Table 4, compared with the control group, the diet supplemented with purslane polysaccharide significantly reduced the diarrhea rate in weaned piglets (P < 0.05). However, there was no significant difference in diarrhea rate between the two experimental groups containing different purslane polysaccharide dosages. No weaned piglets died during the experimental period.

[0132] Table 4 Effects of purslane polysaccharide on diarrhea rate in weaned piglets

[0133] project control group Experimental group A Experimental group B Diarrhea rate / % <![CDATA[40% A ]]> <![CDATA[20% B ]]> <![CDATA[0 B ]]>

[0134] Note: Data in the same industry with different lowercase letters in the shoulder indicate significant differences, different uppercase letters in the shoulder indicate extremely significant differences, and no shoulder or the same shoulder indicate insignificant differences.

[0135] The above examples are provided to those skilled in the art to fully disclose and describe how to implement and use the claimed embodiments, rather than to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. A method for preparing purslane polysaccharide, characterized in that: The following steps are involved: S1. Pretreatment of purslane: taking dried purslane, crushing it to obtain purslane powder, and setting aside; S2. Degreasing treatment: adding the purslane powder to petroleum ether and subjecting to Soxhlet reflux extraction for 3 to 4 hours; after the reflux extraction, taking out the sample, washing it with water, and drying it to obtain defatted purslane powder; S3. Extraction of crude purslane polysaccharide: Mix defatted purslane powder with water and boil for 1.5 to 2 hours; then perform ultrasonic-assisted extraction; after the ultrasonic-assisted extraction is completed, stir for 15 to 20 minutes; filter, and the filtrate is the crude purslane polysaccharide solution; S4. Removal of alcohol-soluble impurities: The crude purslane polysaccharide solution is concentrated, anhydrous ethanol or ethanol solution is added to make the ethanol concentration 75% to 85%, and the solution is allowed to stand for alcohol precipitation; Centrifuge and collect the precipitate; S5, pigment removal: dissolve the precipitate in step S4 in water, add AB-8000 macroporous resin, stir at 35-50°C for 4-5 hours for decolorization; filter to obtain a decolorized solution; S6. Protein removal: Add Sevag reagent to the decolorizing solution and stir for 30 to 50 minutes to perform deproteinization. Let the solution stand to separate the layers, and the lower layer is the deproteinized solution. Repeat the deproteinization process 6 to 8 times. S7. Removal of small molecule impurities: dialyze the deproteinized solution in an 8000-14000 KDa dialysis bag; after dialysis, freeze-dry to obtain crude purslane polysaccharide; S8. Isolation and purification of purslane polysaccharide: dissolve the crude purslane polysaccharide in water, load it onto a DEAE-52 cellulose anion exchange column and elute it with a NaCl solution, collect the eluate of 0.4 M NaCl solution; dialyze the eluate in an 8000-14000 KDa dialysis bag; after dialysis, freeze-dry to obtain purslane polysaccharide.

2. The method for preparing purslane polysaccharide according to claim 1, characterized in that: In step S1, the dried purslane is obtained by the following method: taking fresh whole purslane, washing it, draining the water, and drying it; Or / and: in step S1, the powder is crushed and then passed through a 200-mesh sieve; Or / and: in step S2, the material-liquid ratio is 1:20; Or / and: In step S3, the material-liquid ratio of defatted purslane powder to water is 1:

30.

3. The method for preparing purslane polysaccharide according to claim 1, wherein: In step S3, the parameters of ultrasound-assisted extraction are: temperature 55°C to 60°C, power 400W, and time 40 to 50 minutes; Or / and: in step S3, the extraction is repeated three times, and the filtrates from the three times are combined to obtain a crude purslane polysaccharide solution.

4. The method for preparing purslane polysaccharide according to claim 1, wherein: In step S4, the specific method of standing alcohol precipitation is: standing at 4° C. for 8 to 12 hours; or / and, treating the precipitate as follows: washing the precipitate with 90% ethanol solution, anhydrous ethanol, and acetone in sequence, washing each for 1 to 3 times, and drying.

5. The method for preparing purslane polysaccharide according to claim 1, wherein: In step S5, the mass ratio of crude purslane polysaccharide to water is 1:200, and the amount of AB-8000 macroporous resin added is 60 g / L.

6. The method for preparing purslane polysaccharide according to claim 1, characterized in that: In step S6, the volume ratio of the decolorizing solution to the Sevag reagent is 5:

1.

7. Purslane polysaccharide prepared by the method for preparing purslane polysaccharide according to any one of claims 1 to 6.

8. Use of the purslane polysaccharide according to claim 7 as / in the preparation of a feed additive having a growth-promoting effect and / or a diarrhea-preventing effect on piglets.

9. The use according to claim 8, characterized in that: In specific application, purslane polysaccharide is added to the basic feed of piglets at a ratio of 0.05% to 0.35%.

10. The use according to claim 9, characterized in that: The added ratio of the purslane polysaccharide is 0.1% or 0.3%.

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