Application of houttuynia cordata polysaccharide extract in feed capable of improving intestinal health of piglets

Abstract

The invention discloses application of a herba houttuyniae polysaccharide extract in a feed capable of improving intestinal health of piglets, the feed comprises the herba houttuyniae polysaccharide extract and a basic feed, and the application method of the herba houttuyniae polysaccharide extract comprises the following steps: adding the herba houttuyniae polysaccharide extract into the basic feed, and continuously feeding 21-28-day-old weaned piglets for 28 days, the intestinal flora structure of weaned piglets is adjusted through the houttuynia cordata polysaccharide extract, short-chain fatty acid secretion is promoted, and the intestinal morphology is improved, so that diarrhea prevention and intestinal health improvement are achieved; the herba houttuyniae polysaccharide extract is prepared from the following components in percentage by mass: 20 percent to 21.6 percent of herba houttuyniae polysaccharide, 5 percent to 10 percent of volatile oil, 25 percent to 35 percent of flavonoid, 10 percent to 15 percent of total amino acid, 3.1 percent to 6.1 percent of alkaloid, 3 percent to 10 percent of organic acid and 1.5 percent to 5.5 percent of sterol, and the herba houttuyniae polysaccharide is prepared from glucose, galactose, mannose, arabinose and xylose. The invention aims to improve the survival rate of early weaned piglets, shorten the breeding cycle of a farm and improve the reproductive performance.

Description

Technical Field

[0001] This invention relates to the field of animal husbandry technology, and in particular to the application of a Houttuynia cordata polysaccharide extract in feed that can improve the intestinal health of piglets. 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 act on the intestinal mucosal barrier, leading to weaning diarrhea. 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 causing early weaning diarrhea in piglets. These pathogenic bacteria act on intestinal mucosal epithelial cells by invading or producing toxins, and exacerbate inflammation by affecting the intestinal mucosal immune barrier.

[0003] An existing invention application with publication number CN117562185A discloses a diet for improving the growth performance of piglets and its preparation method. It adds kinase-slow-release microspheres as an acidifier to the weaning diet of 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 shows improvement in growth performance and diarrhea rate, the data itself has large variability. In addition, the diarrhea rate is based on fecal score (0-3 points), which is a subjective judgment and is prone to observer bias. Therefore, there is an urgent need to propose a diet that can stably improve the intestinal health of piglets. Summary of the Invention

[0004] The main objective of this invention is to provide an application of Houttuynia cordata polysaccharide extract in feed that can improve the intestinal health of piglets, aiming to solve the technical problem of poor stability of the existing feed's effect on improving the intestinal health of piglets.

[0005] To achieve the above objectives, this invention provides the application of Houttuynia cordata polysaccharide extract in a diet that improves the intestinal health of piglets. The diet comprises Houttuynia cordata polysaccharide and a basal diet, with the mass ratio of Houttuynia cordata polysaccharide extract to basal diet being (200-800):1000. The application method involves adding Houttuynia cordata polysaccharide extract to the basal diet and feeding it continuously to weaned piglets aged 21-28 days for 28 days. This aims to prevent diarrhea and improve intestinal health by regulating the intestinal flora structure, promoting the secretion of short-chain fatty acids, and improving intestinal morphology of weaned piglets through the Houttuynia cordata polysaccharide extract. The Houttuynia cordata polysaccharide extract comprises 20%-21.6% Houttuynia cordata polysaccharide, 5%-10% volatile oils, 25%-35% flavonoids, 10%-15% total amino acids, 3.1%-6.1% alkaloids, 3%-10% organic acids, and 1.5%-5.5% sterols by mass fraction. The crude polysaccharide from Houttuynia cordata comprises glucose, galactose, mannose, arabinose, and xylose, with the corresponding molar percentages of glucose, galactose, mannose, arabinose, and xylose being 59.80%~60.87%, 20%~21.13%, 8.10%~10.16%, 5.18%~7.28%, and 2%~3.56%, respectively.

[0006] Optionally, the preparation steps of the houttuynia cordata polysaccharide extract are as follows:

[0007] Step 11: Remove impurities from fresh houttuynia cordata rhizomes, dry them, and pulverize them through a 60-mesh sieve to obtain rhizome powder;

[0008] Step 12: Prepare a solution of rhizome powder by mixing it with liquid at a ratio of 1g:(30-40)ml. Then heat the solution to 80-90℃, cool it to 70℃ and reflux it in a water bath for 2-3 hours. Filter the solution to obtain the residue and supernatant.

[0009] Step 13: The residue obtained in step 12 is extracted 2-3 times with the same material-to-liquid ratio, and the supernatants are combined to obtain a combined liquid.

[0010] Step 14: Concentrate the combined liquid to 1 / 10 of the original volume, add 3-5 times the volume of 95% ethanol for precipitation overnight, centrifuge to collect the precipitate and dry it to obtain the crude extract of Houttuynia cordata polysaccharide.

[0011] Optionally, the mass ratio of Houttuynia cordata polysaccharide extract to basal diet is 400:1000.

[0012] Optionally, the regulation of the intestinal flora structure includes increasing the relative abundance of Firmicutes (1-1.41%), Bacteroidetes (17.67-550%), Mucor (98.9-100%), Filamentobacteria (60-66.7%), Clostridium (4.9-122.5%), Prevotella (20-21.1%), Eubacterium (24-25%), Bacillus (99.1-100%), and Broutella (35-40%), and decreasing the relative abundance of Actinobacteria (60-62.8%), Bacteriophages (11-12.5%), Proteobacteria (22.7-36.4%), Spirochetes (22.7-46.7%), and Escherichia coli (94-95.6%).

[0013] Optionally, the improvement in short-chain fatty acid secretion is manifested as an increase in acetic acid content in feces and intestinal contents by 20.58-60%.

[0014] Optionally, the improvement in intestinal morphology includes a 15.97-31.59% reduction in jejunal crypt depth and an 8.11-26.35% increase in the jejunal-chorionic crypt ratio.

[0015] Optionally, the houttuynia cordata polysaccharide extract is added to the basal diet in the form of powder or premix.

[0016] Optionally, in step 12, the material-to-liquid ratio is 1g:40ml.

[0017] Optionally, in step 14, 4 times the volume of 95% ethanol is added for precipitation overnight.

[0018] Beneficial effects:

[0019] This invention provides the application of Houttuynia cordata polysaccharide extract in a diet that improves the intestinal health of piglets. The diet includes the Houttuynia cordata polysaccharide extract and a basal diet. The application method involves adding the Houttuynia cordata polysaccharide extract to the basal diet and feeding it to weaned piglets aged 21-28 days for 28 consecutive days. This aims to prevent diarrhea and improve intestinal health by regulating the intestinal flora structure, promoting short-chain fatty acid secretion, and improving intestinal morphology through the Houttuynia cordata polysaccharide extract. Furthermore, it enhances the growth performance of weaned piglets, increases serum calcium and phosphorus levels, promotes intestinal nutrient digestion, improves intestinal development function, and mitigates intestinal damage caused by weaning stress. It also improves the survival rate of early-weaned piglets, shortens the breeding cycle of farms, and enhances reproductive performance, thereby improving economic benefits and market competitiveness. This method has broad application prospects, low cost, no side effects, and is simple and easy to implement. Attached Figure Description

[0020] Figure 1 shows the separation and purification of HP-D1N1 from Houttuynia cordata polysaccharide. (A) Flowchart of HP-D1N1 separation and purification; (B) Elution curve of HP-D1N1 ion purification; (C) Gel chromatography separation curve of HP-D1N1.

[0021] Figure 2 The ion chromatogram of the monosaccharide composition standard of Houttuynia cordata polysaccharide.

[0022] Figure 3 This is an ion chromatogram of Houttuynia cordata polysaccharide sample.

[0023] Figure 4 shows the methylation analysis of HP-D1N1 in Houttuynia cordata polysaccharide. (A) Gas chromatogram of HP-D1N1 methylation; (B) t-Glc(p); (C) t-Gal(p); (D) 4-Man(p); (E) 4-Gal(p); (F) 4-Glc(p); (G) 4,6-Gal(p).

[0024] Figure 5 shows the structural analysis of HP-D1N1 polysaccharide in Houttuynia cordata polysaccharide. (A) 1H NMR spectrum; (B) 13C NMR spectrum; (C) HSQC analysis; (D) HMBC analysis; (E) NOESY analysis; (F) Schematic diagram of HP-D1N1 structure.

[0025] Figure 6 shows the structural features of HP-D1N1 under a scanning electron microscope. (A) Scanning electron microscope image of HP-D1N1 (10000×); (B) Scanning electron microscope image of HP-D1N1 (5000×); (C) Scanning electron microscope image of HP-D1N1 (2000×); (D) Scanning electron microscope image of HP-D1N1 (1000×).

[0026] Figure 7 The results of the short-chain fatty acid content in the feces of weaned piglets at different ages are shown in the figure. (A) Short-chain fatty acid content in feces on day 1; (B) Short-chain fatty acid content in feces on day 14; (C) Short-chain fatty acid content in feces on day 21.

[0027] Figure 8 The following graphs show the results of short-chain fatty acid content in the contents of different intestinal segments: (A) Short-chain fatty acid content in ileum; (B) Short-chain fatty acid content in cecum; (C) Short-chain fatty acid content in colon; (D) Short-chain fatty acid content in rectum.

[0028] Figure 9The diagram shows the alpha diversity analysis of gut microbiota at the genus level in the contents of the ileum, cecum, and colon. Control represents the control group, and YXC represents the Houttuynia cordata polysaccharide experimental group. Alpha diversity analysis of gut microbiota in the ileum (AD), cecum (EH), and colon (IL) is also presented.

[0029] Figure 10 Figures showing the genus-level β-diversity analysis of gut microbiota in the contents of the ileum, cecum, and colon, where Control represents the control group and YXC represents the Houttuynia cordata polysaccharide experimental group; (AB) genus-level β-diversity analysis of fecal microbiota on day 7; (CD) genus-level β-diversity analysis of fecal microbiota on day 14; (EF) genus-level β-diversity analysis of fecal microbiota on day 21.

[0030] 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

[0031] It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0032] 1. Source, preparation and composition of Houttuynia cordata polysaccharides

[0033] 1.1 Source of Houttuynia cordata polysaccharides

[0034] Houttuynia cordata Thunb., listed in the Chinese Pharmacopoeia, is the fresh whole herb and underground rhizome of the plant *Houttuynia cordata*, belonging to the Saururaceae family. It is a perennial herb, also known as Zhe'ergen, Jicai, and Ziji, and is an important Chinese medicinal and edible herb. The main components of Houttuynia cordata are volatile oils, flavonoids, polysaccharides, and alkaloids. Houttuynia cordata has a long history of use in my country, possessing properties such as clearing heat and detoxifying, reducing swelling and draining pus, and promoting urination. In addition, Houttuynia cordata polysaccharides exhibit significant pharmacological activities in anti-inflammatory, antiviral, antitumor, immunomodulatory, antioxidant, and antibacterial effects. The polysaccharides used in this study are crude polysaccharides extracted from the rhizomes of the Houttuynia cordata plant.

[0035] 1.2 Preparation of Houttuynia cordata polysaccharide

[0036] Remove impurities from fresh houttuynia cordata rhizomes, dry them, and pulverize them through a 60-mesh sieve. Accurately weigh a certain amount of houttuynia cordata and add it to ultrapure water at a material-to-liquid ratio of 1g:(30-40)ml. Heat the mixture to a gentle boil (80-90℃), then reflux it in a 70℃ water bath for 2-3 hours. Filter the houttuynia cordata rhizomes through a gauze bag, collect the supernatant, and repeat the extraction process on the precipitated residue. The two extracts were combined, filtered to remove residue, and the filtrate was concentrated to 1 / 10 of its original volume by rotary evaporation (70℃, -0.085 MPa). 3-5 times the volume of 95% ethanol was added for alcohol precipitation overnight. On the second day, when a large amount of alcohol precipitate was obtained, the precipitate was centrifuged at 4000 rpm for 15 min, the supernatant was discarded, and the precipitate was collected and dried to obtain the Houttuynia cordata polysaccharide extract. This extract contains 20%-21.6% Houttuynia cordata polysaccharides, 5%-10% volatile oils, 25%-35% flavonoids, 10%-15% total amino acids, 3.1%-6.1% alkaloids, 3%-10% organic acids, and 1.5%-5.5% sterols.

[0037] 1.3 Isolation and purification of Houttuynia cordata polysaccharides

[0038] A suitable amount of crude polysaccharide sample was dissolved in pure water to prepare a polysaccharide mother liquor of a certain concentration. The solution was centrifuged at 10000g for 10 min, and the supernatant was purified by passing it through an ion exchange column at a flow rate of 4 ml / min. A gradient elution was performed sequentially using pure water, 0.1M, 0.2M, and 0.3M NaCl solutions, collecting 15 ml tubes at a time, and collecting all eluents. The total sugar content of the eluents in each collection tube was determined using the sulfuric acid-phenol method, and an ion purification elution curve was plotted. The eluents from different fractions were concentrated, dialyzed, and dried to obtain different fractions, designated HP-D1 and HP-D2. HP-D1 was then dissolved in pure water and centrifuged at 10000×g for 10 min. The supernatant was purified by passing it through a Sephacryl S-400HR gel chromatography column at a flow rate of 1 ml / min; 1.5 column volumes of pure water were used for elution to obtain the polysaccharide.

[0039] Separation and purification flowchart as follows Figure 1A As shown. A certain mass of Houttuynia cordata was used as raw material, and crude polysaccharide (38g) was obtained through water extraction and alcohol precipitation. After impurity removal, the crude polysaccharide content was 4.86g. Purification was performed using an ion exchange column (…). Figure 1B Separation and purification using Sephacryl S-400HR gel chromatography column ( Figure 1C The main polysaccharide HP-D1N1 was obtained.

[0040] 1.4 Monosaccharide composition of Houttuynia cordata polysaccharides

[0041] The components of the crude extract of Houttuynia cordata polysaccharide prepared above were determined by analyzing and detecting the monosaccharide components using an electrochemical detector, preferably an ion chromatography system.Figure 2 and 3 The figures show the ion chromatograms of the standard and the sample. The horizontal axis represents the retention time of the detection, and the vertical axis represents the response value of the ion detection. As can be seen from the comparison in Figures 1-2, the Houttuynia cordata polysaccharide includes glucose, galactose, mannose, arabinose, and xylose, with corresponding molar percentages of 59.80%–60.87%, 20%–21.13%, 8.10%–10.16%, 5.18%–7.28%, and 2%–3.56%, respectively.

[0042] 1.5 Uniformity and molecular weight of Houttuynia cordata polysaccharides

[0043] Molecular weight was determined using size exclusion chromatography columns: Ohpak SB-805 HQ (300×8 mm) and Ohpak SB-803 HQ (300×8 mm) in series. The column temperature was 45℃, the injection volume was 100 μL, the mobile phase was A (0.02% NaN3, 0.1M NaNO3), the flow rate was 0.6 mL / min, and the elution gradient was isocratic for 75 min. The concentration of HP-D1N1 was 1 mg / mL, with each injection being 100 μL.

[0044]

[0045] As shown in Table 1, the number-average molecular weight (Mn) of HP-D1N1 is 115.696 kDa, the weight-average molecular weight (Mw) is 247.809 kDa, and the Mw / Mn ratio is 2.142. These data indicate that its molecular weight distribution has high dispersibility and good uniformity.

[0046] 1.6 Methylation Analysis of Houttuynia cordata Polysaccharides

[0047] Take a small amount of sample (2-3 mg), dissolve it in 500 μl DMSO; add 1 mg NaOH and incubate for 30 min; add 50 μl iodomethane solution and react for 1 h; add 1 ml water and 2 ml dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase; repeat the washing with water 3 times, then take the lower dichloromethane phase and dry it with nitrogen; add 100 μl 2M TFA, react at 121℃ for 90 min, and evaporate to dryness at 30℃; add 50 μl 2M ammonia and 50 μl 1M NaBD4, mix well, and react at room temperature for 2.5 h; add 20 μl acetic acid to terminate the reaction, dry with nitrogen, wash twice with 250 μl methanol, and dry with nitrogen; add 250 μl acetic anhydride, vortex to mix, and react at 100℃ for 2.5 h. Add 1 ml of water and let stand for 10 min; add 500 μl of dichloromethane, vortex to mix, centrifuge, discard the aqueous phase, and repeat the water washing 3 times; take the lower dichloromethane phase and analyze it by GC-MS.

[0048]

[0049] To determine the glycosidic bond linkage of HP-D1N1, it was subjected to three methylation treatments to obtain a fully methylated polysaccharide. Six significant peaks were observed from the total ion chromatogram. Figure 4A ), respectively corresponding to the non-reducing ends of glucose (t-Glc(p)) Figure 4B ), non-reducing ends of galactose (t-Gal(p)) Figure 4C ), 4-Man(p) linker of mannose (4-Man(p)) Figure 4D ), galactose 4-link (4-Gal(p)) Figure 4E ), the 4-link of glucose (4-Glc(p)) Figure 4F ) and the 4,6 linker of galactose (4,6-Gal(p)) Figure 4G The quantitative values ​​of specific sugar residues are shown in Table 2. The results indicate that HP-D1N1 has 12 linkage types: t-Man(p), t-Glc(p), t-Gal(p), 5-Ara(f), 4-Man(p), 4-Gal(p), 4-Glc(p), 6-Gal(p), 3,4-Gal(p), 2,4-Man(p), 4,6-Gal(p), and 3,6-Gal(p). Among the various sugar residues, 4-Glc(p) has the largest proportion, indicating that it is likely the main backbone of HP-D1N1. Furthermore, in methylation analysis, HP-D1N1 contains 65.26% glucose, 21.76% galactose, 11.93% mannose, and 1.06% arabinose. This ratio is approximately close to the monosaccharide composition data.

[0050] 1.7 Nuclear Magnetic Resonance (NMR) Spectroscopic Analysis of Houttuynia cordata Polysaccharides

[0051] Dissolve an appropriate amount of purified polysaccharide thoroughly in D2O to prepare a polysaccharide solution with a concentration greater than or equal to 40 mg / mL. Transfer the dissolved solution to an NMR tube, adding 0.5 mL. Place the NMR tube in a 500 MHz NMR spectrometer (Bruck, Germany) and scan the one-dimensional 1H and 13C spectra, and the two-dimensional COSY, HSQC, HMBC, and NOESY spectra at 25 °C. Based on these six spectra, the structure of the conventional polysaccharide is obtained.

[0052]

[0053] from Figure 5AAs can be seen, the polysaccharide signal in 1H NMR is mainly concentrated between δ 3.0 and 5.5 ppm. Multiple coupled signal peaks were identified in the 4.3-5.4 ppm anodic signal region, indicating the presence of various sugar residues in this sample, with corresponding anodic hydrogen chemical shifts of δ 4.4, 4.53, 4.87, 5.11, and 5.3, respectively. Typically, the anodic hydrogen signal of the β-glycosidic configuration is mainly distributed between δ 4.3 and 4.8 ppm, while that of the α-glycosidic configuration is mainly distributed between δ 4.8 and 5.8 ppm. Based on the figure, it is preliminarily determined that HP-D1N1 exhibits a β-glycosidic conformation.

[0054] To obtain comprehensive information from the methylation analysis, we used 13C NMR spectroscopy (…). Figure 5B ) and HSQC spectrum ( Figure 5C The cross-peaks in the anodic region precisely assigned chemical shift signals to specific glycoside residues, identifying the anodic signals present in the sample as: δ 4.53 / 95.85, 5.3 / 99.71, 4.4 / 102.71, 5.11 / 92.03, and 4.87 / 98.11 ppm, respectively denoted as sugar residues A, B, C, D, and E. The HMBC spectrum shows that... Figure 5D The C1 and H4 peaks of sugar residue A show a cross-peak at δ 95.85 / 3.9 ppm, and the C1 and H4 peaks of sugar residue E show a cross-peak at δ 98.11 / 3.9 ppm. This can be seen from the NOESY spectrum. Figure 5E The H1 and H4 of sugar residue A have a cross peak at δ 4.53 / 3.9 ppm; the H1 of sugar residue A has a cross peak at δ 4.53 / 3.71 ppm; the H1 of sugar residue A has a cross peak at δ 4.53 / 4.09 ppm; the H1 of sugar residue B has a cross peak at δ 5.3 / 3.9 ppm; and the H1 of sugar residue C has a cross peak at δ 4.4 / 3.63 ppm.

[0055] Therefore, based on the analysis of one-dimensional and two-dimensional NMR information and methylation results (Table 3), it is inferred that the polysaccharide is mainly composed of interconnected main chains such as →4,6)-α-D-Galp-(1→, →4)-2-OMe-β-D-Glcp-(1→, →4)-α-D-Manp-(1→ and →4)-α-D-Galp, with the side chains mainly composed of β-D-Glcp-(1→ linked to the O-6 position of the sugar residue →4,6)-α-D-Galp-(1→). Figure 5F It consists of ) and others.

[0056] 1.8 Scanning electron microscopy analysis of Houttuynia cordata polysaccharides

[0057] The polysaccharide sample was passed through a 100-mesh sieve, and a small amount was placed on a conductive carbon tape. After gold sputtering, the surface morphology of HP-D1N1 was observed using a high-resolution field emission scanning electron microscope (Zeiss Merlin Compact). Operating conditions: accelerating voltage 10 kV; magnification: 500~10,000.

[0058] The structural features of HP-D1N1 were observed using scanning electron microscopy. Specifically, HP-D1N1 was scanned at 1000×, 2000×, 5000×, and 10000×, and the results are shown below. Figure 6A As shown in Figure -D, under magnification conditions, the surface of the polysaccharide sample HP-D1N1 was observed to be rough and exhibited an irregular fragment structure.

[0059] Furthermore, the present invention provides the application of Houttuynia cordata polysaccharide in the preparation of weaned piglet feed, the method comprising the following steps:

[0060] Step 1: Add Houttuynia cordata polysaccharide extract to the basal diet. The Houttuynia cordata polysaccharide extract comprises 20%–21.6% Houttuynia cordata polysaccharides, 5%–10% volatile oils, 25%–35% flavonoids, 10%–15% total amino acids, 3.1%–6.1% alkaloids, 3%–10% organic acids, and 1.5%–5.5% sterols by mass. The mass ratio of the Houttuynia cordata polysaccharide extract to the basal diet is (200–800):1000. The Houttuynia cordata polysaccharide includes glucose... The product contains glucose, galactose, mannose, arabinose, and xylose, wherein the corresponding molar percentages of glucose, galactose, mannose, arabinose, and xylose are 59.80%~60.87%, 20%~21.13%, 8.10%~10.16%, 5.18%~7.28%, and 2%~3.56%, respectively; preferably, the corresponding molar percentages of glucose, galactose, mannose, arabinose, and xylose are 60.87%, 21.13%, 9.16%, 6.28%, and 2.56%, respectively.

[0061] Step 2: Feed weaned piglets aged 21-28 days continuously for 28 days to prevent diarrhea and improve intestinal health by regulating the intestinal flora structure, promoting the secretion of short-chain fatty acids and improving intestinal morphology through Houttuynia cordata polysaccharide extract. Specifically, regulating the gut microbiota structure includes increasing the relative abundance of Firmicutes (1-1.41%), Bacteroidetes (17.67-550%), Mucor (98.9-100%), Filamentobacteria (60-66.7%), Clostridium (4.9-122.5%), Prevotella (20-21.1%), Eubacterium (24-25%), Bacteroides (99.1-100%), and Broutella (35-40%), while decreasing the relative abundance of Actinobacteria (60-62.8%), Caudata (11-12.5%), Proteobacteria (22.7-36.4%), Spirochetes (22.7-46.7%), and Escherichia coli (94-95.6%). Improved short-chain fatty acid secretion is manifested by a 20.58-60% increase in acetic acid content in feces and intestinal contents. The improvements in intestinal morphology include a 15.9710-31.5925% reduction in jejunal crypt depth and an 8.11-26.35% increase in the jejunal chorioretinoid ratio.

[0062] Furthermore, to better illustrate the effects of the above-mentioned Houttuynia cordata polysaccharide extract on diarrhea and improvement of intestinal health in weaned piglets, specific experimental results are presented below.

[0063] 2. Selection of experimental animals and design of experimental groups

[0064] The experimental subjects selected in this application were 96 three-way crossbred weaned piglets, all 21 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.

[0065] Control group: fed a basal diet;

[0066] YXC-L group: fed with basal diet + 200mg / kg of Houttuynia cordata polysaccharide extract;

[0067] YXC-M group: fed with basal diet + 400mg / kg of Houttuynia cordata polysaccharide extract;

[0068] YXC-H group: fed with basal diet + 800mg / kg of Houttuynia cordata polysaccharide extract;

[0069] The feeding period was 28 days, and other feeding conditions for the experimental animals were conducted according to the standard conditions of a modern breeding farm. Then, the effect experimental data were measured.

[0070] 3. Piglet feed and management

[0071] 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 shown 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 carried out according to the farm's standard immunization program. The pigsty was cleaned twice daily to maintain a clean and tidy environment, and the pens were disinfected every two weeks.

[0072]

[0073] 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. 2) Nutrient levels are calculated values.

[0074] 4. Effects of Houttuynia cordata polysaccharides on growth performance and diarrhea rate in weaned piglets

[0075] Diarrhea data were recorded for the four groups of experimental animals during the feeding experiment. On days 0 and 28 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.

[0076] Average daily weight gain (ADG, kg) = (final weight at the end of the trial - initial weight) / number of days in the trial;

[0077] Average daily feed intake (ADFI, kg) = (Weekly initial feed weight - Weekly final feed weight) / 7;

[0078] Feed conversion ratio (F / G) = Average daily feed intake (kg) / Average daily weight gain (kg);

[0079] Diarrhea rate (%) = 100 × total number of piglets with diarrhea / (total number of piglets in the experiment × total number of days in the experiment).

[0080] The specific experimental results are shown in Table 5:

[0081]

[0082] Data with no letter or the same letter in the same column heading indicates statistical significance (P>0.05), while different lowercase letters indicate statistical significance (P<0.05). The same applies to the following table.

[0083] As shown in Table 5, each dose of Houttuynia cordata polysaccharide extract significantly reduced the diarrhea score and diarrhea rate in piglets (P<0.05), with the 200 mg / kg Houttuynia cordata polysaccharide extract showing the best effect.

[0084] 5. Effects of Houttuynia cordata polysaccharide extract on serum biochemical indicators of weaned piglets

[0085] At the end of the experiment, blood was collected from six piglets in each group. A serum biochemical analyzer was used to detect nine serum biochemical indicators, including calcium (Ca), phosphorus (P), glucose (GLU), aspartate transaminase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total cholesterol (TC), triacylglycerol (TG), and lactate dehydrogenase (LDH). These are shown in Table 6.

[0086]

[0087] As shown in Table 6, the addition of Houttuynia cordata polysaccharide extract can increase the calcium and phosphorus content in serum, especially the 800 mg / kg Houttuynia cordata polysaccharide extract, which significantly increases the calcium and phosphorus content in serum (P<0.05). In this way, feeding Houttuynia cordata polysaccharide extract can improve the immunity of weaned piglets.

[0088] 6. Effects of Houttuynia cordata polysaccharide extract on the morphology of intestinal tissue in weaned piglets

[0089] On day 28 of the experiment, after weighing and recording, a slaughter experiment was conducted. During slaughter, the abdominal cavity was opened, and intestinal segments were quickly removed and separated. Approximately 3 cm segments of the duodenum, jejunum, ileum, and mid-colon were taken and fixed with 4% fixative for preparing intestinal tissue sections. The tissue samples were prepared into paraffin blocks through a process of tissue washing, dehydration, clearing, paraffin embedding, etc. Paraffin sections were then prepared using a LEICARM2135 microtome, followed by spreading, staining, and mounting to create tissue sections. Images were acquired using a photomicrograph, and then software was used to measure villus length and crypt depth, calculating the ratio of villus length to crypt depth for each group.

[0090] The results are shown in Table 7:

[0091]

[0092] As shown in Table 7, the jejunal crypt depth was significantly decreased (P<0.05), while the jejunal villus-to-crypt ratio was increased (p>0.05). No significant differences were observed in duodenal villus height, crypt depth, villus-to-crypt ratio, or ileal villus height, crypt depth, and villus-to-crypt ratio between the dosage group and the control group (p>0.05). Therefore, improving jejunal crypt depth and villus-to-crypt ratio in weaned piglets through intestinal morphology can promote healthy intestinal development and enhance their digestive capacity.

[0093] 7. Effects of Houttuynia cordata polysaccharide extract on the content of short-chain fatty acids in feces and intestinal contents of weaned piglets

[0094] Fresh fecal samples were collected from the 400 mg / kg treatment group at 1, 7, 14 and 21 days of age, and fecal samples from 4 pigs in 6 replicates were collected to determine the short-chain fatty acid content in the fecal samples of weaned piglets in the 400 mg / kg group.

[0095] Accurately weigh 1g of fresh sample into a 10mL centrifuge tube, add 5mL of ultrapure water, vortex, shake and mix for 30min, incubate overnight at 4℃, centrifuge at 10000rpm for 10min, transfer the supernatant, add 4mL of ultrapure water to the precipitate, shake and mix for 30min, centrifuge at 12000rpm for 15min, transfer the supernatant, add it to a 2mL centrifuge tube at a ratio of v:v=9:1 (900ul supernatant + 100ul 25% metaphosphoric acid), mix well, let stand at room temperature for 3-4h, centrifuge, filter, and add to the test vial for analysis.

[0096] Chromatographic conditions: A DB-FFAP column (30m x 250μm x 0.25μm) was used; the carrier gas was high-purity nitrogen (99.999%) at a flow rate of 0.8 mL / min; the auxiliary gas was high-purity hydrogen (99.999%); the detector (FID) temperature was 280℃; the injection port temperature was 250℃; the split ratio was 50:1; the injection volume was 1 μl; the temperature program was as follows: initial temperature 60℃, increased to 220℃ at a rate of 20℃ / min, and held for 1 min. Results are as follows: Figure 7 , 8 As shown.

[0097] 7.1 Short-chain fatty acid content in feces of weaned piglets of different ages

[0098] like Figure 7As shown in Figures A and B, where C represents the control group and YXC represents the Houttuynia cordata polysaccharide experimental group; F1, F2, and F3 represent days 7, 14, and 21, respectively. Compared with the control group, weaned piglets supplemented with 400 mg / kg Houttuynia cordata polysaccharide showed significantly increased levels of acetic acid, propionic acid, and butyric acid in their feces on days 7 and 14 (p < 0.05), while the levels of isobutyric acid, valeric acid, and isovaleric acid showed no significant difference (p > 0.05). This indicates that feeding weaned piglets with Houttuynia cordata polysaccharide can protect the intestinal mucosal barrier, reduce intestinal inflammation, and maintain intestinal homeostasis. Figure 7 As shown in Figure C, compared with the control group, the acetic acid content in weaned piglets supplemented with 400 mg / kg Houttuynia cordata polysaccharide was significantly increased on day 21 (p < 0.05), while the content of other SCFAs showed no significant difference (p > 0.05). This indicates that the addition of Houttuynia cordata polysaccharide extract can significantly increase the acetic acid content in the feces and intestinal contents of weaned piglets, promoting intestinal digestion and absorption.

[0099] 7.2 Short-chain fatty acid content in the contents of different intestinal segments

[0100] exist Figure 8 In the diagram, C represents the control group, YXC represents the Houttuynia cordata polysaccharide experimental group; H, M, J, and Z represent the ileum, cecum, colon, and rectum, respectively. Figure 8 As shown in A and B, compared with the control group, the content of acetic acid in the ileal contents of weaned piglets supplemented with 400 mg / kg Houttuynia cordata polysaccharide extract was significantly reduced (p < 0.05), while the content of SCFAs in the cecal contents was not significantly different (p > 0.05).

[0101] like Figure 8 As shown in C and D, compared with the control group, the content of acetic acid in the rectal contents of weaned piglets supplemented with 400 mg / kg Houttuynia cordata polysaccharide extract was significantly increased (p < 0.05), while the content of SCFAs in the colon contents was not significantly different (p > 0.05).

[0102] 8. Effects of Houttuynia cordata polysaccharide extract on the microbial composition of the ileum, cecum and colon contents of weaned piglets.

[0103] On day 28 of the feeding experiment, six well-fitting, similarly sized weaned piglets were selected from each group. Immediately after slaughter, the contents of the ileum, cecum, and colon were collected, and metagenomic sequencing of the microbial species within these contents was performed. Using the Illumina NovaSeq sequencing platform, small fragment libraries were constructed and sequenced using paired-end sequencing. Species composition was analyzed by reading splicing and filtering, clustering or denoising, and species annotation and abundance analysis. The results are shown in Tables 8-9.

[0104]

[0105] As shown in Table 8, compared with the control group, at the phylum level, the Houttuynia cordata polysaccharide group in the ileum contents increased the relative abundance of Firmicutes, Bacteroidetes, and Mucor, and decreased the relative abundance of Actinobacteria and Caudata phages, but the differences were not significant (P>0.05); the Houttuynia cordata polysaccharide group in the cecal contents increased the relative abundance of Bacteroidetes, and decreased the relative abundance of Proteobacteria, Caudata phages, and Spirochetes, but the differences were not significant (P>0.05); the Houttuynia cordata polysaccharide group in the colon contents increased the relative abundance of Bacteroidetes and Filamentobacteria, and decreased the relative abundance of Proteobacteria and Spirochetes, but the differences were not significant (P>0.05). Specifically, it increases the relative abundance of Firmicutes (1-1.41%), Bacteroidetes (17.67-550%), Mucor (98.9-100%), and Filamentobacteria (60-66.7%), while decreasing the relative abundance of Actinobacteria (60-62.8%), Caudata (11-12.5%), Proteobacteria (22.7-36.4%), and Spirochetes (22.7-46.7%).

[0106]

[0107] As shown in Table 9, compared with the control group, at the genus level, the Houttuynia cordata polysaccharide group in the ileum contents increased the relative abundance of Clostridium and decreased the relative abundance of Escherichia coli, but the difference was not significant (P>0.05); the Houttuynia cordata polysaccharide group in the cecal contents increased the relative abundance of Prevotella, Clostridium, and Eubacterium, and decreased the relative abundance of Rochezoctonia and Ruminococcus, but the difference was not significant (P>0.05); the Houttuynia cordata polysaccharide group in the colon contents increased the relative abundance of Bacillus faecalis (P<0.05), Prevotella, Clostridium, and Broutella, and decreased the relative abundance of Rochezoctonia, but the difference was not significant (P>0.05). Specifically, it increases the relative abundance of Clostridium spp. by 4.9-122.5%, Prevotella spp. by 20-21.1%, Eubacterium spp. by 24-25%, Bacillus spp. by 99.1-100%, and Broutella spp. by 35-40%, while decreasing the relative abundance of Escherichia coli by 94-95.6%.

[0108] 9. Effects of Houttuynia cordata polysaccharide extract on α-diversity of gut microbiota in weaned piglets

[0109] Specifically, the analysis of the α-diversity of gut microbiota in weaned piglets by Houttuynia cordata polysaccharide extract is shown in the figure below. Figure 9 As shown, compared with the control group, the ileal contents microbial indices of Chao1, Ace, Shannon, and Simpson were all increased in the Houttuynia cordata polysaccharide group. Figure 9 AD). The cecal contents showed an increasing trend in Chao1 and Ace indices, but the increase was not significant. Figure 9EF), while the Shannon and Simpson indices decreased ( Figure 9 GH). The colon contents microbiota Chao1 and Ace indices decreased ( Figure 9 IJ), while both the Shannon and Simpson indices rose (IJ). Figure 9 KL). The results showed that Houttuynia cordata polysaccharide extract helped increase the richness and diversity of gut microbiota in the foregut of weaned piglets, while reducing the richness of gut microbiota.

[0110] 10. Effects of Houttuynia cordata polysaccharide extract on β-diversity of gut microbiota in weaned piglets

[0111] Specifically, the analysis of the β-diversity of gut microbiota in weaned piglets by Houttuynia cordata polysaccharide extract is shown in the figure below. Figure 10 As shown, principal coordinate analysis (PCoA) based on the Bray_curtis algorithm showed no significant differences between the foregut and hindgut groups. Figure 10 A, C, E), which is consistent with the results of Non-Metric Multidimensional Scaling (NMDS) analysis. Figure 10 (B, D, F) indicates that there are differences in microorganisms between the control group and the Houttuynia cordata polysaccharide group, but these differences are not significant.

[0112] The above experimental results indicate that feeding weaned piglets with Houttuynia cordata polysaccharide extract significantly increases the levels of calcium and phosphorus in serum and acetic acid in feces and intestinal contents; it also increases the ratio of jejunal villus height to crypt depth and significantly reduces jejunal crypt depth, promoting intestinal nutrient digestion, improving intestinal development and function, and mitigating intestinal damage caused by weaning stress. Feeding with Houttuynia cordata polysaccharide extract also significantly reduces the diarrhea rate, exhibiting a significant anti-diarrheal effect on piglets, possibly by increasing the abundance of beneficial bacteria in the intestinal flora, thereby regulating intestinal flora homeostasis and reducing diarrhea in weaned piglets.

[0113] Furthermore, based on the above effects, Houttuynia cordata polysaccharide extract can also be added to feed as an additive. Generally, the feed additive is a powder or premix, and the carrier is selected from at least one of malt, hawthorn, and glucose.

[0114] Furthermore, Houttuynia cordata polysaccharide extract can be used as the main raw material for drugs that improve the intestinal immune function of weaned piglets, and Houttuynia cordata polysaccharide extract can be used as the main raw material for drugs that promote the intestinal development of weaned piglets, with a certain amount of enteric-coated preparation added to the corresponding drugs.

[0115] 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. The application of a Houttuynia cordata polysaccharide extract in a diet that improves the intestinal health of piglets, characterized in that, The feed includes Houttuynia cordata polysaccharide extract and basal feed, wherein the mass ratio of Houttuynia cordata polysaccharide extract to basal feed is (200-800):1000; The method of application is to add Houttuynia cordata polysaccharide extract to the basic diet and feed it to weaned piglets aged 21-28 days for 28 days. The purpose is to prevent diarrhea and improve intestinal health by regulating the intestinal flora structure, promoting the secretion of short-chain fatty acids and improving intestinal morphology of weaned piglets through Houttuynia cordata polysaccharide extract. The Houttuynia cordata polysaccharide extract comprises 20%–21.6% Houttuynia cordata polysaccharides, 5%–10% volatile oils, 25%–35% flavonoids, 10%–15% total amino acids, 3.1%–6.1% alkaloids, 3%–10% organic acids, and 1.5%–5.5% sterols by mass. The Houttuynia cordata polysaccharide includes glucose, galactose, mannose, arabinose, and xylose, with corresponding molar percentages of 59.80%–60.87%, 20%–21.13%, 8.10%–10.16%, 5.18%–7.28%, and 2%–3.56%, respectively.

2. The application according to claim 1, characterized in that, The preparation steps of the houttuynia cordata polysaccharide extract are as follows: Step 11: Remove impurities from fresh houttuynia cordata rhizomes, dry them, and pulverize them through a 60-mesh sieve to obtain rhizome powder; Step 12: Prepare a solution of rhizome powder by mixing it with liquid at a ratio of 1g:(30-40)ml. Then heat the solution to 80-90℃, cool it to 70℃ and reflux it in a water bath for 2-3 hours. Filter the solution to obtain the residue and supernatant. Step 13: The residue obtained in step 12 is extracted 2-3 times with the same material-to-liquid ratio, and the supernatants are combined to obtain a combined liquid. Step 14: Concentrate the combined liquid to 1 / 10 of the original volume, add 3-5 times the volume of 95% ethanol for precipitation overnight, centrifuge to collect the precipitate and dry it to obtain the crude extract of Houttuynia cordata polysaccharide.

3. The application according to claim 1, characterized in that, The mass ratio of Houttuynia cordata polysaccharide extract to basal diet was 400:1000.

4. The application according to any one of claims 1-3, characterized in that, The regulation of intestinal flora structure includes increasing the relative abundance of Firmicutes (1-1.41%), Bacteroidetes (17.67-550%), Mucor (98.9-100%), Filamentobacteria (60-66.7%), Clostridium (4.9-122.5%), Prevotella (20-21.1%), Eubacterium (24-25%), Bacillus (99.1-100%), and Broutella (35-40%), and decreasing the relative abundance of Actinobacteria (60-62.8%), Bacteriophages (11-12.5%), Proteobacteria (22.7-36.4%), Spirochetes (22.7-46.7%), and Escherichia coli (94-95.6%).

5. The application according to any one of claims 1-3, characterized in that, The improved secretion of short-chain fatty acids was manifested by an increase of 20.58-60% in the acetic acid content of feces and intestinal contents.

6. The application according to any one of claims 1-3, characterized in that, The improvements in intestinal morphology include a 15.97-31.59% reduction in jejunal crypt depth and an 8.11-26.35% increase in the jejunal chorioretinoid ratio.

7. The application according to any one of claims 1-3, characterized in that, The houttuynia cordata polysaccharide extract is added to the basal diet in powder or premix form.

8. The application according to claim 2, characterized in that, In step 12, the material-to-liquid ratio is 1g:40ml.

9. The application according to claim 2, characterized in that, In step 14, add 4 times the volume of 95% ethanol and precipitate overnight.

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