Siberian solomonseal rhizome polysaccharide extract weaned piglet feed additive and application thereof

Abstract

The invention provides a rhizoma polygonati polysaccharide extract weaned piglet feed additive and application thereof.The feed additive comprises a rhizoma polygonati polysaccharide extract, and the rhizoma polygonati polysaccharide extract comprises, by mass, 36%-49.8% of rhizoma polygonati polysaccharide, 3%-12% of saponin, 2%-8% of flavone, 30%-45% of total amino acid and 0.2%-1% of alkaloid. Polygonatum sibiricum polysaccharide has various pharmacological activities such as anti-inflammation and bacteriostasis, immunity regulation, tumor resistance, oxidation resistance, blood sugar and blood fat reduction and the like, the polygonatum sibiricum polysaccharide extract is reasonably added into weaned piglet basic feed breeding, and the application dosage of the polygonatum sibiricum polysaccharide extract is 150-600 mg per kg of weaned piglet basic feed; the feed antibiotic can replace feed antibiotics, and can effectively reduce the diarrhea rate of weaned piglets, regulate the intestinal flora structure and improve the abdominal and intestinal morphology of the weaned piglets, so that the survival rate of the weaned piglets in the early stage is increased, the breeding cycle of a farm is shortened, and the reproductive performance is improved.

Description

Technical Field

[0001] This invention relates to the field of animal husbandry technology, and in particular to a feed additive for weaned piglets made from Polygonatum polysaccharide extract and its application. Background Technology

[0002] With the continuous improvement of breeding techniques, the weaning age of piglets has gradually decreased from 60 days to 35 days or even earlier, greatly increasing the number of piglets produced by sows annually. However, due to improper early weaning or inadequate feeding and management after early weaning, piglets are highly susceptible to diarrhea in the early weaning period, leading to a decline in economic benefits. Currently, the causes of diarrhea in piglets are broadly classified into two categories: non-infectious diarrhea and infectious diarrhea. Non-infectious diarrhea mainly includes factors such as the incomplete physiological functions of piglets, inadequate feeding and management, and stress responses. Infectious diarrhea includes bacterial diarrhea, viral diarrhea, and diarrhea caused by parasites. Meanwhile, early weaning can also disrupt the gut microbiota structure of piglets. Pathogenic bacteria in the gut can trigger weaning diarrhea by acting on the intestinal mucosal barrier. The gut microbiota formed in suckling piglets is predominantly composed of anaerobic bacilli. Early weaning stress induces a loss of gut microbiota diversity, altering the relative abundance of bacteria. The number of anaerobic bacilli such as lactobacilli decreases, while the number of facultative anaerobic bacteria such as Escherichia coli increases. Pathogenic Escherichia coli is the main pathogen inducing early weaning diarrhea in piglets. Pathogenic bacteria in the gut act on intestinal mucosal epithelial cells by invading or producing toxins, and exacerbate inflammation by affecting the intestinal mucosal immune barrier. In the past, feed antibiotics were widely used to prevent weaning diarrhea and promote growth, but their overuse has led to increased bacterial resistance, threatening human health. Currently, many countries worldwide have completely banned the use of feed antibiotics. Therefore, developing safe and effective alternatives (such as plant extracts) to regulate gut microbiota and enhance mucosal barrier function has become a key research direction for solving the problem of early weaning diarrhea.

[0003] The invention application with publication number CN117562185A discloses a feed for improving the growth performance of piglets and its preparation method. It adds kinase-releasing microspheres as an acidifier to the weaning feed for piglets. Kinase is a composite acidifier containing phosphoric acid, lactic acid, citric acid and fumaric acid. Anhydrous calcium chloride is used as a crosslinking agent. Chitosan and carboxymethyl cellulose are used to prepare hydrogel microspheres by coagulation method to encapsulate kinase. Although it can improve the pH environment of the piglet's stomach, the preparation is relatively complicated and the cost is high. Therefore, a high-cost-performance feed for piglets that can be used for weaning is proposed. Summary of the Invention

[0004] The main objective of this invention is to provide a feed additive for weaned piglets made from Polygonatum polysaccharide extract and its application, aiming to solve the technical problem of low cost-effectiveness of existing feeds.

[0005] To achieve the above objectives, the present invention provides a feed additive for weaned piglets containing Polygonatum polysaccharide extract. The feed additive includes Polygonatum polysaccharide extract, which comprises 36% to 49.8% Polygonatum polysaccharide by mass, 3% to 12% saponins, 2% to 8% flavonoids, 30% to 45% total amino acids, and 0.2% to 1% alkaloids.

[0006] Optionally, the preparation method of the Polygonatum polysaccharide extract includes the following steps:

[0007] Step 11: Cut the Polygonatum rhizome into 1-3mm thin slices and soak them in water at a ratio of 1g:(16-21)ml until the core is thoroughly soaked.

[0008] Step 12: Continue heating to 80-90℃ and simmer for 2-3 hours. Filter to obtain the residue and filtrate.

[0009] Step 13: Continue to boil the residue obtained in step 12 2-3 times with the same material-to-liquid ratio, filter and combine all the filtrates to obtain a combined liquid;

[0010] Step 14: Concentrate the combined liquid under reduced pressure to a relative density of 1.16-1.20, add 96% ethanol for 10-12 hours for precipitation, centrifuge to collect the precipitate and vacuum microwave dry to obtain crude Polygonatum polysaccharide.

[0011] Step 15: Purification of crude polysaccharide. The crude polysaccharide extract solid was dissolved in pure water and then enzymatically hydrolyzed overnight with papain. The upper aqueous phase was collected by adding 1 / 4 volume of chloroform and n-butanol, the lower aqueous phase was collected by adding petroleum ether, and the lower aqueous phase was adsorbed overnight with macroporous resin AB-8. After collecting the liquid, it was dialyzed with a 3000 Da dialysis bag for 48 hours to remove small molecule components. The polysaccharide solution was then precipitated with alcohol and dried to obtain Polygonatum polysaccharide.

[0012] Step 16: Purification of Polygonatum polysaccharide ions. First, dissolve an appropriate amount of crude polysaccharide sample in pure water to prepare a polysaccharide mother liquor of a certain concentration. Then, centrifuge the mother liquor at 10000g for 10 min. Take the supernatant and purify it by passing it through an ion exchange column at a flow rate of 4 ml / min. Elute sequentially with pure water, 0.1M, 0.2M, and 0.3M NaCl solutions, collecting 15 ml of each solution as one tube and retaining all eluent. Then, determine the total sugar content of the eluent from each collection tube using the sulfuric acid-phenol method, and plot the ion purification elution curve. Select and confirm the elution peak (the main peak is selected by default). Then, combine the eluents from each collection tube corresponding to the same elution peak, concentrate them to 1 / 5 of the original volume by rotary evaporation, and then dialyze them with a 3000 Da dialysis bag for 48-72 h for desalting. After freeze-drying, accurately weigh a small amount of polysaccharide solid using the sulfuric acid-phenol method, dissolve it in water, and add sulfuric acid-phenol solution. The phenol reagent was reacted in the dark for 10 minutes, and the absorbance was measured at 490 nm to identify the content and purity of the polysaccharide after ion purification.

[0013] Step 17: Gel purification of Polygonatum polysaccharide. Take the polysaccharide sample after ion purification, add pure water to prepare a polysaccharide stock solution of a certain concentration, centrifuge at 10000g for 10 min, and take the supernatant to separate and purify by passing it through a gel chromatography column at a flow rate of 1 ml / min. Elute with pure water at 1.5 column volumes, collecting one tube per 10 ml, and collect all eluents. Determine the total sugar content of the eluent in each collection tube using the sulfuric acid-phenol method, and plot the gel purification elution curve accordingly. Select and confirm the elution peak (default is the main peak). Combine the eluents from each collection tube corresponding to the same elution peak, concentrate them to 1 / 5 of the original volume by rotary evaporation, and then freeze-dry. Identify the content and purity of the gel-purified polysaccharide using the sulfuric acid-phenol method. Specifically, accurately weigh a small amount of polysaccharide solid, dissolve it in water, add sulfuric acid-phenol reagent, react in the dark for 10 min, and measure the absorbance at 490 nm.

[0014] Optionally, in step 11, the material-to-liquid ratio is 1g:16ml.

[0015] Optionally, in step 14, the combined liquid is concentrated under reduced pressure to a relative density of 1.16.

[0016] Optionally, the Polygonatum polysaccharide comprises glucose and fructose, wherein the corresponding molar percentages of glucose and fructose are 6.37% and 93.63%, respectively.

[0017] Optionally, the dosage of the added ingredient is 150-600 mg of Polygonatum polysaccharide extract per kg of weaned piglet basal diet.

[0018] In addition, to achieve the above objectives, the present invention also provides the application of Polygonatum polysaccharide extract as a feed additive for weaned piglets.

[0019] Optionally, the application is in the preparation of a feed additive that reduces the rate of diarrhea in weaned piglets.

[0020] Optionally, the application is in the preparation of feed additives that regulate the gut microbiota structure of weaned piglets.

[0021] Optionally, the application is in the preparation of a feed additive to improve the morphology of the abdominal intestine of weaned piglets.

[0022] Beneficial effects:

[0023] This invention provides a Polygonatum sibiricum polysaccharide extract feed additive for weaned piglets and its application. The application involves adding the Polygonatum sibiricum polysaccharide extract to a basal diet and feeding it to 28-day-old weaned piglets for 28 consecutive days. This effectively regulates the intestinal flora structure and improves intestinal morphology, thereby reducing diarrhea rates. The Polygonatum sibiricum polysaccharide extract comprises 36%–49.8% Polygonatum sibiricum polysaccharides, 3%–12% saponins, 2%–8% flavonoids, 30%–45% total amino acids, and 0.2%–1% alkaloids. The mass ratio of the Polygonatum sibiricum polysaccharide extract to the basal diet is (150–600):1000. The monosaccharide composition of the Polygonatum sibiricum polysaccharide includes glucose and fructose, with corresponding molar percentages of 6.37% and 93.63%, respectively. This improves the growth performance and serum antioxidant capacity of weaned piglets, promotes intestinal nutrient digestion, and improves intestinal development and intestinal microbiota. It increases the survival rate of weaned piglets, shortens the breeding cycle of farms, and enhances economic value and market competitiveness. This method has broad application prospects, low cost, no side effects, and is simple and easy to implement. Attached Figure Description

[0024] Figure 1 (A) Polygonatum polysaccharide extract eluted on DEAE-FF column; (B) Polygonatum polysaccharide eluted on Sephacryl S-400HR column; (C) Polygonatum polysaccharide extraction and purification process; (D) Ion chromatogram of standard; (E) Ion chromatogram of Polygonatum monosaccharide composition.

[0025] Figure 2 Scanning electron microscope (SEM) images of Polygonatum polysaccharide (A) PSP (5000×); (B) SEM image of Polygonatum polysaccharide (10000×);

[0026] Figure 3. Absolute molecular weight and methylation results of Polygonatum polysaccharides (2M-121℃-1.5h). (A) Absolute molecular weight analysis of Polygonatum polysaccharides; (B) Total ion chromatogram of Polygonatum polysaccharides; (C, D) PMAAs mass spectra of β-D-Fruf-(2→); (E, F) PMAAs mass spectra of β-D-Fruf-(2→); (G) PMAAs mass spectra of α-D-Glcp-(1→); (H, I) PMAAs mass spectra of β-D-Fruf-(2→).

[0027] Figure 4. NMR spectra of Polygonatum polysaccharide: (A) 1H NMR spectrum; (B) 13C NMR spectrum; (C) COSY analysis; (D) NOESY analysis; (E) HSQC analysis; (F) HMBC analysis; (G) DEPT-135 analysis; (H) Schematic diagram of PSP structure.

[0028] Figure 5 The graph shows the antioxidant effects of Polygonatum polysaccharide extract on serum of weaned piglets. Low dose represents group A of Polygonatum polysaccharide experiment, medium dose represents group B of Polygonatum polysaccharide experiment, and high dose represents group C of Polygonatum polysaccharide experiment.

[0029] Figure 6 The effect of adding Polygonatum polysaccharide extract to the diet on the alpha diversity of gut microbiota in weaned piglets;

[0030] Figure 7 Principal component analysis of the gut microbiota at the genus level of adding Polygonatum polysaccharide extract to the diet of weaned piglets.

[0031] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0032] It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0033] 1. Source, preparation and composition of Polygonatum polysaccharide extract

[0034] 1.1 Source of Polygonatum polysaccharide extract

[0035] Polygonatum is a perennial herbaceous plant belonging to the Liliaceae family. Based on its shape, it is classified into Polygonatum sibiricum, Polygonatum multiflorum, and Polygonatum yunnanense. Polygonatum sibiricum has a sweet taste and neutral properties, and enters the spleen, lung, and kidney meridians. It has the effects of tonifying qi and nourishing yin, strengthening the spleen, moistening the lungs, and benefiting the kidneys. Clinically, it is mainly used to treat symptoms such as lung deficiency and dryness, spleen and stomach disharmony, fatigue, and insufficient essence and blood, and is known as the "King of Tonifying Blood and Qi." Polygonatum sibiricum possesses various pharmacological activities, such as anti-inflammatory and antibacterial effects, immune regulation, anti-tumor effects, antioxidant effects, and hypoglycemic and hypolipidemic effects. The Polygonatum sibiricum polysaccharide in this study is a polysaccharide extract from Polygonatum sibiricum.

[0036] 1.2 Preparation of Polygonatum polysaccharide extract

[0037] Take a certain amount of Polygonatum rhizome, cut it into 1-3 mm thin slices, and soak it in water at a material-to-liquid ratio of 1g:16ml to 1g:21ml until the core is clear. Add ultrapure water, heat to a gentle boil (80-90℃), and decoct for 2-3 hours. Filter the Polygonatum rhizome through a gauze bag, filter the extract, and collect the filtrate for later use. Add 16 times the amount of water to the filtered Polygonatum rhizome residue and repeat 2-3 times. Combine the filtrates obtained from multiple filtrations and concentrate the filtrate under reduced pressure to a relative density of 1.16-1.20 (80℃). The concentrated liquid was placed in an alcohol precipitation tank, and 96% alcohol was added to a final concentration of 80%. The mixture was allowed to stand at 4℃–104℃ for at least 12 hours for alcohol precipitation. The precipitate was then collected by centrifugation at 4600 rpm for 10 minutes. The alcohol was recovered from the supernatant, and the precipitate was identified as a Polygonatum polysaccharide extract. This extract comprised 36%–49.8% Polygonatum polysaccharides, 3%–12% saponins, 2%–8% flavonoids, 30%–45% total amino acids, and 0.2%–1% alkaloids by mass. The highest polysaccharide mass ratio in the Polygonatum polysaccharide extract was 49.8%.

[0038] 1.3 Extraction, purification and monosaccharide composition of Polygonatum polysaccharides

[0039] Using 5 kg of Polygonatum sibiricum as raw material, a polysaccharide extract (552 g, yield 11%) was obtained from the raw material by water extraction and alcohol precipitation. The extract was then purified to remove impurities. Finally, it was fractionated and eluted on a DEAE Seplife FF anion exchange column. Figure 1A The eluent in the tube was collected for further purification using gel permeation chromatography on a Sephacryl S-400HR gel chromatography column. The eluent in the tube was then freeze-dried. Figure 1B The extracted polysaccharide (84.0% purity) was designated as Polygonatum sibiricum polysaccharide and used for subsequent structural analysis and activity studies. The extraction and purification flowchart of Polygonatum sibiricum polysaccharide is shown below. Figure 1C As shown in the figure. The polysaccharide was broken down into monosaccharides by acid hydrolysis, and the monosaccharides were separated using an ion-exchange chromatography column to determine the monosaccharide composition of PSP. The monosaccharide composition of PSP was analyzed by comparing the chromatograms with those of standard monosaccharides; specifically, the content and composition of monosaccharides were determined using an electrochemical detector. The final results are shown in the figure. Figure 1D As shown in Figures E, Polygonatum polysaccharides are mainly heteropolysaccharides composed of fructose (Fru) and glucose (Glc), with molar percentages of 93.63% and 6.37%, respectively.

[0040] 1.4 Electron Microscopy Analysis of Polygonatum Polysaccharides

[0041] like Figure 2The results show that under magnification of 5K and 10K, Polygonatum polysaccharide exhibits an irregular and rough appearance, with spherical structures of varying particle sizes attached to it.

[0042] 1.5 Molecular weight analysis of Polygonatum polysaccharides

[0043] Table 1 lists the molecular weight parameters (Mn, Mp, Mw, and Mz). The Mn, Mp, Mw, and Mz of Polygonatum polysaccharide are 4.723 kDa, 4.615 kDa, 4.806 kDa, and 4.912 kDa, respectively. The polydispersity (Mw / Mn) ratio is 1.018, indicating that Polygonatum polysaccharide has a narrow molecular weight distribution.

[0044]

[0045] 1.6 Homogeneity and Methylation Analysis of Polygonatum Polysaccharides

[0046] like Figure 3A The results show that Polygonatum polysaccharides exhibit uniform and symmetrical peaks, indicating homogeneity. Individual sugar components of the polysaccharide were derivatized into partially methylated sugar alcohol acetates (PMAAs), and analyzed and quantified by GC-MS. The results indicate that we obtained the total ion chromatogram of Polygonatum polysaccharides (PMAAs). Figure 3B Seven peaks were identified in the study. Figure 3C ,D,E,F,G,H,I). These peaks correspond to four PMAAs: 2,5-di-O-acetyl-(2-deuterio)-1,3,4,6-tetra-O-methyl hexitols (mannitol, glucitol), 1,2,5-tri-O-acetyl-(2-deuterio)-3,4,6-tri-O-methylhexitols (mannitol,glucitol), 1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl glucitol and 1,2,5,6-tetra-O-acetyl-(2-deuterio)-3,4-di-O-methyl hexitol (mannitol,glucitol). The corresponding glycosidic bonds were determined to be β-D-Fruf-(2→、→1)-β-D-Fruf-(2→、→6)-α-D-Glcp-(1→ and →1,6)-β-D-Fruf-(2→, and the corresponding molar ratios are summarized in Table 2.

[0047]

[0048] 1.7 Nuclear Magnetic Resonance Analysis of Polygonatum Polysaccharides

[0049] To further elucidate the structural characteristics of Polygonatum polysaccharide, NMR spectroscopy analysis was performed. No significant absorption peaks were detected in the δ 4.4–5.3 ppm range (with the δ 4.71 ppm peak being the HOD solvent peak), consistent with the fructose signal, indicating the presence of a fructose structure in Polygonatum polysaccharide. A weak absorption peak at δ 5.32 ppm was identified, belonging to the α-configuration of glucose, denoted as sugar residue D. Non-anomeric hydrogen signals were mainly concentrated in the δ 3.1–4.2 ppm region, with some signals showing significant overlap. Figure 4A It also needs to be combined with COSY spectrum (). Figure 4C ) and HSQC spectrum ( Figure 4E The H2-H6 chemical shifts of each sugar residue were assigned.

[0050] Three coupling signal peaks were mainly identified in the anodic carbon region of Polygonatum polysaccharide, with chemical shifts of δ 103.96, 103.16, and δ 103.78 ppm, respectively, denoted as sugar residues A, B, and C. The cross-peaks in the anodic region were analyzed using 1H NMR and HSQC spectra. Figure 4A , Figure 4E The anodic signal of sugar residue D was determined to be δ5.32 / 92.1ppm; sugar residues A, B and C showed no cross-peaks in the anodic region of the HSQC spectrum, consistent with the structural characteristics of fructose [5]. Combined with DEPT-135 and 13C NMR spectra ( Figure 4B , Figure 4G The methylene (δ 60.74, 60.43, 59.91, 62.22, 62.46, 63.11 ppm) signals were obtained. Based on the analysis of the methylation results of Polygonatum polysaccharide, the anolyte signal, and the literature reports, it was inferred that sugar residue A is →1)-β-D-Fruf-(2→[6], residue B is β-D-Fruf-(2→[6], residue C is →1,6)-β-D-Fruf-(2→[7], and residue D is →6)-α-D-Glcp-(1→[7]). The 1H and 13C chemical shifts were assigned, and the results are shown in Table 3.

[0051] Based on the chemical shifts of the 13C and 1H residues of each sugar residue in Polygonatum polysaccharide, combined with HMBC and NOESY spectra ( Figure 4F , Figure 4DThe structure and linkages of the polysaccharide were analyzed. According to the HMBC spectrum, there was a cross-peak between C2 and H1 of sugar residue A (δ 103.96 / 3.78(3.82) ppm); and a cross-peak between C2 and H1 of sugar residue C (δ 103.96 / 3.57(3.66) ppm). A cross-peak between C2 and H6 of sugar residue B (δ 103.16 / 3.85 ppm) was observed. A cross-peak between C2 and H1 of sugar residue A (δ 103.78 / 3.78(3.82) ppm) was observed. Due to the low content of sugar residue D, a cross-peak between H1 of sugar residue D and H1 of sugar residue C was only found in the NOESY spectrum (δ 5.32 / 3.66 ppm), and no related signals with other sugar residues were found in the HMBC spectrum.

[0052] Therefore, by combining one-dimensional and two-dimensional NMR information with methylation results, the possible putative structure of Polygonatum polysaccharide was deduced ( Figure 4H The main chain of Polygonatum polysaccharide is mainly composed of →1)-β-D-Fruf-(2→ and →1,6)-β-D-Fruf-(2→ linked together. The side chains are mainly composed of β-D-Fruf-(2→ linked to the O-6 position of →1,6)-β-D-Fruf-(2→), and there are a very small amount of →6)-α-D-Glcp-(1→ linked to the O-1 position of →1,6)-β-D-Fruf-(2→).

[0053]

[0054] Furthermore, the present invention provides the application of Polygonatum polysaccharide extract in the preparation of a feed additive that reduces the rate of diarrhea in weaned piglets, the method comprising the following steps:

[0055] Step 1: Add Polygonatum polysaccharide extract to the basal diet. The Polygonatum polysaccharide extract comprises 36%–49.8% Polygonatum polysaccharide, 1%–10% saponins, 0.1%–1% flavonoids, 5%–15% total amino acids, and 0.01%–0.1% alkaloids by mass. The mass ratio of the Polygonatum polysaccharide extract to the basal diet is (150–600):1000. The monosaccharide composition of the Polygonatum polysaccharide includes glucose and fructose, with corresponding molar percentages of 6.37% and 93.63%, respectively.

[0056] Step 2: Weaned piglets aged 28 days were fed continuously for 28 days to reduce the diarrhea rate by regulating the gut microbiota structure and improving intestinal morphology. Specifically, regulating the gut microbiota structure included increasing the abundance of Clostridium spp. in the ileum, Lactobacillus in the cecum, and Prevotella in the colon, while decreasing the abundance of Escherichia coli in the ileum. Improving intestinal morphology included a 25.16% reduction in jejunal crypt depth and a 41.89% increase in the jejunal-chorioretinoid ratio.

[0057] Furthermore, to better illustrate the effects of the above-mentioned Polygonatum polysaccharide extract on diarrhea and improvement of intestinal health in weaned piglets, specific experimental results are presented below.

[0058] 2. Selection of experimental animals and design of experimental groups

[0059] The experimental subjects selected in this application were 96 three-way crossbred weaned piglets, all 28 days old, which were randomly divided into 4 groups according to their weight, with 6 replicates in each group and 4 pigs in each replicate.

[0060] Control group: fed a basal diet;

[0061] Polygonatum polysaccharide group A: Feed basal diet + 150mg / kg Polygonatum polysaccharide extract;

[0062] Polygonatum polysaccharide group B: Feed basal diet + 300mg / kg Polygonatum polysaccharide extract;

[0063] Polygonatum polysaccharide group C: fed with basal diet + 600mg / kg of Polygonatum polysaccharide extract;

[0064] The pre-feeding period was 3 days, and the experimental period was 28 days. Other feeding conditions for the experimental animals were carried out according to the routine conditions of a modern breeding farm. Then, the effect experimental data were measured.

[0065] 3. Piglet feed and management

[0066] Based on the NRC (1998) standards for piglet nutritional requirements, a diet formula was developed to meet the nutritional needs of piglets. The diet ingredients and nutrient components are detailed in Table 4. The pig farm was designed as a fully enclosed pigsty with slatted metal pens at the rear and nipple-type water outlets. All treatment groups were fed pelleted feed four times a day at 7:00, 11:00, 14:00, and 18:00, with a feeding standard of 1 / 3 remaining feed in the trough. During the experiment, pigs had free access to feed and water throughout the period, and immunization was performed according to the farm's standard immunization program. The pigsties were cleaned twice daily to maintain a clean and tidy environment, and disinfection was carried out every two weeks.

[0067]

[0068] Note: 1) The following substances are supplied per kilogram of diet: choline chloride 500 mg, vitamin A 10 500 IU, vitamin D3 3 300 IU, vitamin E 30 IU, vitamin K3 3 mg, vitamin B1 3 mg, vitamin B2 7.5 mg, vitamin B6 4.5 mg, vitamin B12 0.007 mg, niacin 30 mg, pantothenate 15 mg, folic acid 1.5 mg, iron 160 mg, biotin 0.12 mg, Cu 10 mg, Fe 150 mg, Zn 135 mg, Mn 15 mg, I 0.5 mg, Se 0.27 mg.

[0069] 2) Nutritional levels are calculated values.

[0070] 4. Effects of Polygonatum polysaccharide extract on growth performance and diarrhea rate in weaned piglets

[0071] Diarrhea data were recorded for the four groups of experimental animals during the feeding experiment. On days 0 and 31 of the experiment, the weight of each piglet was measured to calculate the average daily weight gain of the piglets. Diarrhea was recorded daily for each pig during the experiment.

[0072] Average daily weight gain (ADG) = (final weight - initial weight) / number of days in the trial;

[0073] Diarrhea rate (%) = Number of piglets with diarrhea during the experimental period / (Number of piglets per group × Number of days in the experiment) × 100.

[0074] The experimental results are shown in Table 5:

[0075]

[0076] Note: P<0.01 in the table indicates that the difference is highly significant.

[0077] As shown in Table 5, during the experiment, compared with the control group, the average daily weight gain of piglets fed with Polygonatum polysaccharide at a concentration of 600 mg / kg increased by 300 g / d. The piglets were in good health during the experiment, indicating that feeding Polygonatum polysaccharide can significantly reduce the diarrhea rate of weaned piglets (P<0.01).

[0078] 5. Effects of feeding Polygonatum polysaccharide on serum antioxidant capacity levels in weaned piglets

[0079] On day 28 of the experiment, one piglet close to the average weight was selected from each replicate. After fasting, 4-5 mL of blood was collected from the vena cava using a vacuum blood collection tube without anticoagulant. The blood was left to stand at room temperature for 1-2 hours, then centrifuged at 3500 r / min for 10 min. The serum was then aliquoted into centrifuge tubes and stored at -20℃ for the determination of blood antioxidant indicators.

[0080] Total antioxidant capacity (T-AOC), total glutathione peroxidase (GPx), and malondialdehyde (MDA) were determined according to a specific kit (Beijing Box Biotechnology Co., Ltd., Beijing, China). Results are as follows: Figure 5 As shown.

[0081] like Figure 5 Results: Compared with the control group, Polygonatum polysaccharide B group significantly reduced the MDA content of peroxidase free radical reaction products in the serum of weaned piglets. Polygonatum polysaccharide A and Polygonatum polysaccharide C groups also showed a trend of reducing MDA. Figure 5 B).

[0082] 6. Effects of Polygonatum polysaccharide extract on intestinal morphology of weaned piglets

[0083] At the end of day 28 of the experiment, one piglet with a weight close to the average weight of each replicate was selected. Six piglets were placed in each of the control group, Polygonatum polysaccharide group A, Polygonatum polysaccharide group B, and Polygonatum polysaccharide group C, for a total of 24 pigs, for the slaughter experiment. The selected pigs were fasted for 12 hours before being weighed and their pre-slaughter weight recorded. After exsanguination and slaughter, the intestinal segments were quickly separated. Two to three centimeters of the middle section of the duodenum, jejunum, and ileum were fixed in 4% paraformaldehyde for the determination of changes in intestinal morphology and structure.

[0084] Duodenal, jejunal, and ileal segments were harvested, soaked in physiological saline to remove contents, and then placed in 4% paraformaldehyde fixative. After dehydration, embedding, sectioning, dewaxing, HE staining, clearing, and mounting, the intestinal mucosa morphology and structure were observed and photographed using an optical microscope (n=6). For each section, six intact and straight-oriented villi and crypts were selected for height measurement. The ratio of villus height to crypt depth (V / C) was calculated for each villus and crypt, and the average value was statistically analyzed. The results are shown in Table 6.

[0085]

[0086] Note: P<0.01 in the table indicates that the difference is extremely significant.

[0087] As shown in Table 6, compared with the control group, the Polygonatum polysaccharide C group significantly increased the villus length of the duodenum (p<0.01) and significantly increased the ratio of villus length to crypt depth in both the duodenum and jejunum (p<0.01). Intestinal morphology, including villus height, crypt depth, and the ratio of villus height to crypt depth, collectively reflects the health and absorptive status of intestinal function. Decreased villus height and crypt depth indicate impaired intestinal mucosal function and reduced intestinal digestive and absorptive capacity. Furthermore, it achieved a 25.16% reduction in jejunal crypt depth and a 41.89% increase in the jejunal-villus-crypt ratio. These results indicate that feeding weaned piglets with Polygonatum polysaccharide can significantly improve the intestinal morphology of weaned piglets, thereby improving intestinal health and reducing the occurrence of diarrhea.

[0088] 7. Differences in the composition of intestinal microbiota of weaned piglets by Polygonatum polysaccharide extract.

[0089] At the end of day 28 of the experiment, one piglet with a weight close to the average weight of that replicate was selected from each replicate, and the intestinal contents of the ileum and cecum were collected and immediately frozen in liquid nitrogen. The collected samples were stored at -80°C for subsequent metagenomic sequencing of the intestinal contents.

[0090] Based on the Illumina NovaSeq sequencing platform, small fragment libraries were constructed using paired-end sequencing. Species composition was analyzed by splicing and filtering reads, clustering or denoising, and performing species annotation and abundance analysis. The results are shown in Tables 7-8.

[0091]

[0092] Note: P<0.01 in the table indicates that the difference is extremely significant.

[0093] As shown in Table 7, compared with the control group, at the phylum level, the HJ group of ileal contents showed an increase in the relative abundance of Firmicutes and a decrease in the relative abundance of Proteobacteria and Actinobacteria; the HJ group of cecal contents showed an increase in the relative abundance of Firmicutes and a decrease in the relative abundance of Proteobacteria; the HJ group of colonic contents showed an increase in the relative abundance of Bacteroidetes, but the difference was not significant (P>0.05).

[0094]

[0095] As shown in Table 8, compared with the control group, at the genus level, the HJ group of ileal contents significantly increased the relative abundance of Clostridium (P < 0.05) and decreased the relative abundance of Escherichia coli; the HJ group of cecal contents increased the relative abundance of Lactobacillus and Clostridium; the HJ group of colonic contents significantly increased the relative abundance of Prevotella, but the difference was not significant. These results indicate that dietary supplementation with Polygonatum polysaccharide alters the abundance of intestinal microbiota in weaned piglets (P > 0.05).

[0096] 8. Effects of Polygonatum polysaccharide extract on α-diversity of gut microbiota in weaned piglets

[0097] Compared with the control group, the levels of Ace, Chao1, Shannon, and Simpson indices in the ileum contents of the Polygonatum polysaccharide group were all increased (e.g., ...). Figure 6 A to 6D). Cecum (e.g.) Figure 6 E to 6F) and colon (such as Figure 6 The Ace and Chao1 indices of microorganisms in the contents of piglets from I to 6J were decreased; the Shannon and Simpson indices did not change significantly. The results indicate that Polygonatum polysaccharide extract helps increase the richness and diversity of gut microbiota in the foregut of weaned piglets, while decreasing the richness of gut microbiota.

[0098] 9. Effects of Polygonatum polysaccharide extract on β-diversity of gut microbiota in weaned piglets

[0099] Effects of Polygonatum polysaccharide extract on the beta diversity of gut microbiota in weaned piglets, such as Figure 7 As shown in the figure, in the analysis of ileal microbiota, PC1 explained 56.18% of the variance, and PC2 explained 27.06% of the variance. The samples from the treatment group and the control group showed a certain degree of separation in the figure, indicating that there were differences in the ileal microbial community composition between the two groups. Figure 7 A and Figure 7 B). Regarding the cecal microbiota, PC1 explained 66.96% of the variance, while PC2 explained 18.48%. The sample distribution showed a certain degree of differentiation between the treatment and control groups, indicating that dietary treatment affected the cecal microbiota community structure. Figure 7 C and Figure 7 D). In the analysis of colonic microbiota, PC1 explained as much as 79.46% and 23.82% of the variance, respectively. The figure clearly shows a certain degree of separation in the sample distribution between the treatment group and the control group. Figure 7 E and Figure 7F). In summary, the results of this study indicate that dietary supplementation with Polygonatum polysaccharide extract alters the microbial community structure of the ileum, cecum, and colon of weaned piglets, mainly by increasing the abundance of Clostridium spp. in the ileum, Lactobacillus in the cecum, and Prevotella in the colon, while decreasing the abundance of Escherichia coli in the ileum.

[0100] The above experimental results indicate that feeding weaned piglets with Polygonatum polysaccharide extract significantly reduces the MDA content (a product of peroxidation free radical reaction) in their serum; increases duodenal villus length and significantly increases the ratio of villus length to crypt depth in both the duodenum and jejunum; and significantly improves intestinal morphology, thereby enhancing intestinal health and reducing diarrhea. Furthermore, it can increase the richness and diversity of gut microbiota in the foregut while reducing the richness of hindgut microbiota, thus decreasing diarrhea in weaned piglets.

[0101] Furthermore, while some studies have utilized mouse diarrhea models to develop plant extracts for treating piglet diarrhea, rodent models such as mice are only suitable for preliminary screening of anti-diarrheal candidates. Their gut microbiota structure, immune development patterns, and weaning stress responses differ significantly from those of piglets. For example, the proportion of lactobacilli in the mouse gut (approximately 60%) is much higher than in piglets (approximately 30%), and the colonization and resistance mechanisms of E. coli also differ. More importantly, the pathological changes unique to piglets after weaning, such as villus atrophy and decreased digestive enzyme activity, are difficult to fully replicate in mouse models. Therefore, "anti-diarrheal effects" derived solely from mouse experiments may overestimate or misjudge their practical application potential. The reliability of the aforementioned experiments is based on the fact that all data originated from in vivo experiments in piglets, including microbiota sequencing, intestinal mucosal barrier indicators, and diarrhea rate statistics. These results directly reflect the actual response of the target species, providing a precise basis for the development of alternative products.

[0102] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or system that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or system. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or system that includes that element.

[0103] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0104] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. Any equivalent structural or procedural transformations made based on the description and drawings of the present invention, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A feed additive for weaned piglets made from Polygonatum polysaccharide extract, characterized in that, The feed additive includes Polygonatum polysaccharide extract, which comprises 36% to 49.8% Polygonatum polysaccharide by mass, 3% to 12% saponins, 2% to 8% flavonoids, 30% to 45% total amino acids, and 0.2% to 1% alkaloids.

2. The feed additive according to claim 1, characterized in that, The preparation method of the Polygonatum polysaccharide extract includes the following steps: Step 11: Cut the Polygonatum rhizome into 1-3mm thin slices and soak them in water at a ratio of 1g:(16-21)ml until the core is thoroughly soaked. Step 12: Continue heating to 60°C and simmer for 4 hours. Filter to obtain the residue and filtrate. Step 13: Continue to boil the residue obtained in step 12 three times with the same material-to-liquid ratio, filter and combine all the filtrates to obtain a combined liquid; Step 14: Concentrate the combined liquid under reduced pressure to a relative density of 1.16-1.20, add 95% ethanol for 12 hours for precipitation, centrifuge to collect the precipitate and vacuum microwave dry to obtain Polygonatum polysaccharide extract.

3. The feed additive according to claim 2, characterized in that, In step 11, the material-to-liquid ratio is 1g:16ml.

4. The feed additive according to claim 2, characterized in that, In step 14, the combined liquid is concentrated under reduced pressure to a relative density of 1.

16.

5. The feed additive according to claim 1, characterized in that, The Polygonatum polysaccharide comprises glucose and fructose, with the corresponding molar percentages of glucose and fructose being 6.37% and 93.63%, respectively.

6. The feed additive according to claim 1, characterized in that, The recommended dosage is 150-600 mg of Polygonatum polysaccharide extract per kg of basal diet for weaned piglets.

7. The application of the Polygonatum polysaccharide extract as a feed additive for weaned piglets according to any one of claims 1-6.

8. The application according to claim 7, characterized in that, Specifically, it is used in the preparation of feed additives that reduce the rate of diarrhea in weaned piglets.

9. The application according to claim 7, characterized in that, Specifically, it is used in the preparation of feed additives that regulate the structure of the intestinal flora in weaned piglets.

10. The application according to claim 7, characterized in that, Specifically, it is used in the preparation of feed additives that improve the morphology of the abdominal intestines of weaned piglets.

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