A suspension for improving intestinal toxicity of piglets caused by DON and a preparation method and application thereof

Abstract

The application discloses a suspension for improving DON-caused piglet intestinal toxicity and a preparation method and application thereof, and belongs to the technical field of feed additives, and comprises the following raw materials: hesperidin 40-60 mg / mL, sodium butyrate 50-150 mg / mL and PR-39 antibacterial peptide 10-15 mg / mL. The suspension provided by the application can also regulate intestinal flora balance, promote the proliferation of beneficial bacteria such as lactobacillus, and inhibit the growth of harmful bacteria. The effect of the suspension provided by the application is significantly better than that of single component or partial combination, and the suspension provides an efficient and safe nutritional intervention strategy for preventing and treating animal body damage caused by feed mycotoxin pollution.

Description

Technical Field

[0001] This invention belongs to the field of feed additive technology, and in particular relates to a suspension that improves DON-induced intestinal toxicity in piglets, its preparation method and application. Background Technology

[0002] Deoxynivalenol (DON) is a widespread mycotoxin in agricultural production, mainly produced by pathogenic fungi such as *Fusarium graminearum* and *Fusarium nobile*. It readily contaminates grains and their processing byproducts, posing a direct threat to livestock health. In my country's current mycotoxin contamination landscape, DON has the highest rate of exceeding standards in feed ingredients. Surveys show that by 2025, the detection rate of DON in pig compound feed in my country will reach 100%, with a peak concentration of 3817.5 μg / kg. After piglets ingest DON, the plasma DON concentration rapidly reaches its peak and is quickly distributed to multiple organs. DON first accumulates in the liver and kidneys, and then accumulates in the intestines. Piglets' intestinal systems are immature and highly sensitive to DON. Ingestion of feed containing DON will reduce nutrient absorption, decrease feed conversion ratio, and even cause acute gastrointestinal diseases. Excessive DON exposure causes damage to the small intestinal epithelial barrier, inflammatory infiltration, mitochondrial dysfunction, and may even induce excessive autophagy, necrotizing apoptosis, and other adverse biological processes. Therefore, it is of great significance to conduct in-depth research on the intestinal toxicological effects of DON and propose corresponding intervention measures.

[0003] Hesperidin (HDN) is a dihydroflavonoid compound widely found in the fruits of citrus plants (Citrus spp., Rutaceae family) and possesses various biological activities. Current pharmacological studies indicate that hesperidin exhibits multiple effects, including anti-inflammatory, antioxidant, and fatty acid metabolism-promoting properties, and can effectively alleviate liver and kidney damage caused by DON exposure in animals. However, existing technologies have not yet disclosed the use of hesperidin in synergistic effects with other substances to improve DON-induced intestinal toxicity in piglets.

[0004] Sodium butyrate (NaB) is a sodium salt of a short-chain fatty acid and a primary energy substrate for intestinal epithelial cells. It is widely used in poultry intestinal health care and the prevention and intervention of inflammation. PR-39 antimicrobial peptide is composed of 39 amino acids, rich in proline and arginine, and has potent anti-inflammatory and intestinal protective effects.

[0005] Therefore, how to provide a feed additive that improves DON-induced intestinal toxicity in piglets by combining hesperidin with sodium butyrate and PR-39 antimicrobial peptide is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes a suspension that improves DON-induced intestinal toxicity in piglets, along with its preparation method and application.

[0007] To achieve the above objectives, the present invention provides the following technical solution: A suspension for improving DON-induced enteric toxicity in piglets, comprising the following ingredients: Hesperidin 40-60 mg / mL, sodium butyrate 50-150 mg / mL and PR-39 antimicrobial peptide 10-15 mg / mL.

[0008] Further preferred ingredients include the following: Hesperidin 60 mg / mL, sodium butyrate 150 mg / mL and PR-39 antimicrobial peptide 15 mg / mL.

[0009] A method for preparing a suspension that improves DON-induced enteric toxicity in piglets includes the following steps: Sodium butyrate and hesperidin were homogenized under high pressure in a pH buffer matrix, and then PR-39 antimicrobial peptide was added under stirring. After adjusting the pH, the suspension was obtained.

[0010] Preferably, the pH buffer matrix comprises 0.5% hydroxypropyl methylcellulose and 0.1% Tween-80 by mass.

[0011] Preferably, the solvent for the pH buffer matrix is ​​water, more preferably purified water.

[0012] Preferably, the pH adjustment is to adjust the pH to 7.0.

[0013] Preferably, the pressure of the high-pressure homogenizer is 80 MPa and the temperature is 25°C.

[0014] Preferably, the initial suspension is stirred at a temperature of 25°C, a stirring rate of 12,000 rpm, and a stirring time of 5 min.

[0015] Preferably, the final suspension is stirred at a temperature of 25°C, a stirring rate of 300 rpm, and a stirring time of 8 min.

[0016] The use of a suspension as described above in the preparation of a drug to improve DON-induced intestinal toxicity in piglets.

[0017] Preferably, during the application process, the ratio of the amount of suspension added to the basal diet of piglets is 1 mL: 1 kg.

[0018] Compared with the prior art, the present invention has the following advantages and technical effects: The suspension provided by this invention can significantly improve the weight gain and daily weight gain of DON-infected piglets, restoring growth indicators to near-healthy levels, protecting intestinal morphological integrity, maintaining normal villus height and villus-to-cryptoplasm ratio, and systematically inhibiting DON-induced oxidative stress, apoptosis, and ferroptosis through a multi-target pathway of "antioxidation-antiferroptosis-antiapoptosis." Furthermore, the suspension provided by this invention can regulate intestinal flora balance, promoting the proliferation of beneficial bacteria such as lactobacilli and inhibiting the growth of harmful bacteria. The suspension provided by this invention is significantly more effective than single components or partial combinations, providing an efficient and safe nutritional intervention strategy for controlling DON contamination in feed. Attached Figure Description

[0019] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 The graph shows the relationship between the age and weight of piglets after feeding in the control group, DON model group, comparative examples 1-4 and examples 1-3. Figure 2 A shows the intestinal villi of piglets after feeding with the control group, DON model group, comparative examples 1, 4 and example 3 under an optical microscope; B shows the villi-to-crystal ratio (V / C) of the small intestine of piglets. Figure 3 The results of GSEA analysis of protein in jejunal tissue of piglets after feeding in the DON model group and Example 3 group; Figure 4 The results show the intestinal flora analysis of piglets after feeding the control group, DON model group, and Example 3 group. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0022] Unless otherwise specified, all raw materials used in the embodiments of this invention were purchased through commercial channels; Hesperidin was purchased from Shanghai Yuanye Biotechnology Co., Ltd., CAS No.: 520-26-3, purity: HPLC ≥ 98%; Sodium butyrate was purchased from Shanghai Yuanye Biotechnology Co., Ltd., CAS No.: 156-54-7, purity: HPLC ≥ 98%; PR39 antimicrobial peptide was purchased from Nanjing Taopu Biotechnology Co., Ltd., molecular formula: C 229 H 346 N 70 O 40 Purity: HPLC ≥ 98%; The piglets were healthy weaned three-way crossbred piglets (Duroc × Large White × Landrace), 4 weeks old and of similar weight, and were raised in the experimental animal room of the College of Veterinary Medicine, Northeast Agricultural University.

[0023] Unless otherwise specified, room temperature or normal temperature in the embodiments of the present invention refers to 25±3℃.

[0024] Example 1 A suspension for improving DON-induced enteric toxicity in piglets, comprising the following ingredients: Hesperidin 40 mg / mL, sodium butyrate 50 mg / mL and PR-39 antimicrobial peptide 10 mg / mL.

[0025] A method for preparing a suspension that improves DON-induced enteric toxicity in piglets includes the following steps: A pH buffer matrix containing hydroxypropyl methylcellulose, Tween-80, and a pH buffer solution (0.05 mol / L sodium dihydrogen phosphate-disodium hydrogen phosphate buffer stock solution) was prepared by adding hydroxypropyl methylcellulose (0.5%), Tween-80 (0.1%), and a pH buffer solution (final matrix concentration 0.02 mol / L) to purified water. Sodium butyrate and hesperidin were uniformly dispersed in the pH buffer matrix at 35°C using high-shear dispersion (10000 rpm, 4 min) and high-pressure homogenization (80 MPa) to form a stable initial suspension. The suspension was then stirred at 12000 rpm for 5 min at temperatures below 25°C. PR-39 antimicrobial peptide was added to the initial suspension and gently stirred (300 rpm, 8 min). The volume was adjusted, and the pH was adjusted to 7.0. The final suspension was stored at 2-8°C.

[0026] Example 2 A suspension for improving DON-induced enteric toxicity in piglets, comprising the following ingredients: Hesperidin 50 mg / mL, sodium butyrate 100 mg / mL and PR-39 antimicrobial peptide 12.5 mg / mL.

[0027] A pH buffer matrix containing hydroxypropyl methylcellulose, Tween-80, and a pH buffer solution (0.05 mol / L sodium dihydrogen phosphate-disodium hydrogen phosphate buffer stock solution) was prepared by adding hydroxypropyl methylcellulose (0.5%), Tween-80 (0.1%), and a pH buffer solution (final matrix concentration 0.02 mol / L) to purified water. Sodium butyrate and hesperidin were uniformly dispersed in the pH buffer matrix at 35°C using high-shear dispersion (10000 rpm, 4 min) and high-pressure homogenization (80 MPa) to form a stable initial suspension. The suspension was then stirred at 12000 rpm for 5 min at temperatures below 25°C. PR-39 antimicrobial peptide was added to the initial suspension and gently stirred (300 rpm, 8 min). The volume was adjusted, and the pH was adjusted to 7.0. The final suspension was stored at 2-8°C.

[0028] Example 3 A suspension for improving DON-induced enteric toxicity in piglets, comprising the following ingredients: Hesperidin 60 mg / mL, sodium butyrate 150 mg / mL and PR-39 antimicrobial peptide 15 mg / mL.

[0029] A pH buffer matrix containing hydroxypropyl methylcellulose, Tween-80, and a pH buffer solution (0.05 mol / L sodium dihydrogen phosphate-disodium hydrogen phosphate buffer stock solution) was prepared by adding hydroxypropyl methylcellulose (0.5%), Tween-80 (0.1%), and a pH buffer solution (final matrix concentration 0.02 mol / L) to purified water. Sodium butyrate and hesperidin were uniformly dispersed in the pH buffer matrix at 35°C using high-shear dispersion (10000 rpm, 4 min) and high-pressure homogenization (80 MPa) to form a stable initial suspension. The suspension was then stirred at 12000 rpm for 5 min at temperatures below 25°C. PR-39 antimicrobial peptide was added to the initial suspension and gently stirred (300 rpm, 8 min). The volume was adjusted, and the pH was adjusted to 7.0. The final suspension was stored at 2-8°C.

[0030] Comparative Example 1 A suspension, differing from Example 3 only in that it does not include sodium butyrate and PR-39 antimicrobial peptide, but includes the following ingredient: hesperidin 60 mg / mL.

[0031] A method for preparing a suspension, using the raw materials described above in this comparative example, with other process steps and parameters being the same as in Example 1.

[0032] Comparative Example 2 A suspension, differing from Example 3 only in that it does not include hesperidin and PR-39 antimicrobial peptide, but comprises the following ingredient: sodium butyrate 150 mg / mL.

[0033] A method for preparing a suspension, using the raw materials described above in this comparative example, with other process steps and parameters being the same as in Example 1.

[0034] Comparative Example 3 A suspension, differing from Example 3 only in that it does not include hesperidin and sodium butyrate, but includes the following ingredient: PR39 antimicrobial peptide 15 mg / mL.

[0035] A method for preparing a suspension, using the raw materials described above in this comparative example, with other process steps and parameters being the same as in Example 1.

[0036] Comparative Example 4 A suspension, differing from Example 3 only in that it does not include hesperidin, but comprises the following ingredients: sodium butyrate 150 mg / mL and PR39 antimicrobial peptide 15 mg / mL.

[0037] A method for preparing a suspension, using the raw materials described above in this comparative example, with other process steps and parameters being the same as in Example 1.

[0038] Technical effects: 1. Animal experiments 1) Assessment of the dynamic effects of suspension on the growth performance of DON-infected piglets By continuously monitoring piglet weight gain, daily weight gain, feed intake, and feed conversion ratio, the effects of the hesperidin, sodium butyrate, and PR39 antimicrobial peptide composition described in this invention on the growth performance of DON-infected piglets were dynamically evaluated. From the perspective of weight gain, a key indicator in production practice, the effects of this composition in alleviating DON toxicity and promoting animal growth were directly verified. Preparation of DON experimental diet: Naturally moldy corn was added to the basal diet to achieve a final DON concentration of 1 mg / kg (referring to the Feed Hygiene Standard GB 13078-2017) to obtain the DON experimental diet.

[0039] Seventy-two weaned piglets, 4 weeks old, of similar weight (7.5±0.5 kg), and in good health, were randomly divided into 9 groups of 8 piglets each, and housed in individual pens. The experimental period was 28 days. The experimental grouping was set as follows: 1) Control group: fed with a basal diet (composition shown in Table 1).

[0040] 2) DON model group: fed with DON experimental diet.

[0041] 3) Comparative Example 1 (DON + hesperidin group): The diet was fed with DON experimental diet, and 1 ml of the suspension obtained from Comparative Example 1 was added per kilogram of feed.

[0042] 4) Comparative Example 2 (DON + Sodium Butyrate Group): The diet was fed with DON experimental diet, and 1 ml of the suspension obtained from Comparative Example 2 was added per kilogram of feed.

[0043] 5) Comparative Example 3 (DON+PR-39 group): The diet was fed with DON experimental diet, and 1 ml of the suspension obtained from Comparative Example 3 was added per kilogram of feed.

[0044] 6) Comparative Example 4 (DON + Sodium Butyrate + PR-39 Group): Feed the DON experimental diet, and add 1 ml of the suspension obtained from Comparative Example 4 per kilogram of feed.

[0045] 7) Example 1 group (DON + hesperidin + sodium butyrate + PR39 low dose group): fed with DON experimental diet, 1 ml of the suspension obtained in Example 1 was added per kilogram of feed.

[0046] 8) Example 2 group (DON + hesperidin + sodium butyrate + PR39 medium dose group): fed with DON experimental diet, 1 ml of the suspension obtained in Example 2 was added per kilogram of feed.

[0047] 9) Example 3 group (DON + hesperidin + sodium butyrate + PR39 high dose group): fed with DON experimental diet, 1 ml of the suspension obtained in Example 3 was added per kilogram of feed.

[0048] Weight measurement: On the mornings of day 0 and day 28 of the experiment, all piglets were weighed on an empty stomach, and individual weights were recorded. A calibrated electronic weighbridge with an accuracy of 0.01 kg was used. All weight and growth performance data are expressed as mean ± standard deviation, and statistical analysis was performed using Prism 10 software.

[0049] Table 1. Composition and nutrient levels of basal diet (air-dried basal diet) The results are as follows Figure 1 As shown in Table 2, Table 2: Changes in piglet weight in different treatment groups Note: Data are expressed as mean ± standard deviation.

[0050] The results are as follows Figure 1 As shown in Table 2-4, Table 3: Changes in average daily weight gain of piglets in different treatment groups Note: Data are expressed as mean ± standard deviation.

[0051] Table 4: Changes in average daily feed intake of piglets in different treatment groups Note: Data are expressed as mean ± standard deviation.

[0052] It can be seen that throughout the entire experimental period, the weight of piglets in all groups showed an increasing trend with age, but the rate of increase differed significantly. The control group exhibited the best growth curve, with its weight steadily increasing from approximately (7.45 ± 0.21) kg to approximately (17.00 ± 0.22) kg. The weight gain of piglets in the DON model group was significantly inhibited; from the middle of the experiment, their weight was significantly lower than that of the control group, and by the end of the experiment, their weight was extremely significantly lower than that of the control group. The weight gain curves of Comparative Examples 1, 2, 3, and 4 were between those of the DON model group and the control group. Furthermore, in the later stages of the experiment, the weight of these treatment groups was significantly higher than that of the DON model group, but still significantly lower than that of the control group, indicating that single or partial application of DON can alleviate growth inhibition to some extent. The weight gain curves of Examples 1-3 were close to those of the control group. In the later stages of the experiment, the difference in weight between the group and the DON model group gradually widened. From about 49 days of age, the weight of the group was significantly higher than that of the DON model group and the control groups 1-4. By the end of the experiment, the weight of the group was not statistically different from that of the control group and was extremely significantly higher than that of the DON model group.

[0053] In summary, this part of the experiment, through dynamic monitoring of body weight and calculation of growth performance indicators, confirmed at the overall animal level that DON-contaminated diets can significantly inhibit the growth of piglets, manifested as slow weight gain, decreased daily weight gain and feed intake, and reduced feed conversion efficiency. Specifically, the addition of hesperidin, sodium butyrate, and PR-39 antimicrobial peptide alone, or the combined addition of sodium butyrate and PR39 antimicrobial peptide, can alleviate the growth inhibition caused by DON to some extent, but the effect is limited. However, the triple combination of hesperidin, sodium butyrate, and PR39 antimicrobial peptide provided by this invention can most effectively reverse the negative effects of DON on piglet growth, far exceeding the sum of the technical effects of comparative examples 1-4. It can restore the weight gain curve, daily weight gain, and other key production indicators of DON-treated piglets to levels close to those of normal healthy piglets.

[0054] 2) Protective effect of suspension on intestinal morphology of piglets challenged with DON virus In vivo experiments on piglets verified the effect of the suspension obtained in this invention on alleviating growth inhibition and intestinal structural damage in piglets under natural conditions caused by DON.

[0055] Based on the above experimental setup, medication was administered at regular intervals every morning. After the experiment, three piglets were randomly selected from each replicate, anesthetized, and slaughtered. A 2 cm segment of the midjejunum was immediately collected, rinsed with physiological saline, and a portion was fixed in 4% paraformaldehyde for histological observation. The fixed intestinal tissue was dehydrated, embedded in paraffin, sectioned (5 μm thick), and stained with hematoxylin and eosin (HE). The tissue was then observed and photographed under an optical microscope. For each section, ten intact, straight-oriented villi and their adjacent crypts were randomly selected. Image analysis software was used to measure villi height and crypt depth, and the villi-to-crypt ratio was calculated.

[0056] The results are as follows Figure 2 As shown in Parts A and B, compared with the control group, the control group had intact intestinal villi structure, neat and slender arrangement, clear crypt structure, and a high villus-to-crypt ratio. The DON model group showed severe intestinal morphological damage, manifested as significant villus atrophy, breakage, and shedding, with a significantly reduced height; crypt depth increased compensatorily, leading to a highly significant decrease in the villus-to-crypt ratio (V / C). Comparative Examples 1 and 4 showed some relief of intestinal damage, with partial improvement in villus height and crypt depth, and an increasing trend in the villus-to-crypt ratio compared to the model group, but still lagged behind the control group. The intestinal morphology of Example 3 group was almost completely protected; villus height and crypt depth showed no significant difference from the control group, the villus-to-crypt ratio returned to normal levels, significantly higher than the DON model group, and under the microscope, the villus structure was intact and tightly arranged, forming a stark contrast to the DON model group.

[0057] Animal experiments have shown that feeding piglets a diet contaminated with DON can lead to severe atrophy of jejunal villi and deepening of crypts, among other typical intestinal structural damage. Hesperidin alone, or sodium butyrate in combination with PR39, can alleviate DON-induced growth inhibition and intestinal morphological damage to some extent. However, the triple combination of hesperidin, sodium butyrate, and PR39 antimicrobial peptide provided in Example 3 of this invention exhibits a more superior synergistic protective effect in vivo, exceeding the sum of the effects of Comparative Examples 1 and 4. It can almost completely protect the integrity of the intestinal morphology and maintain normal villus height and villus-crypt ratio. In conclusion, the suspension of hesperidin, sodium butyrate, and PR39 antimicrobial peptide described in this invention can effectively protect against DON-induced intestinal morphological damage in piglets, demonstrating its effectiveness in alleviating DON intestinal toxicity in piglets at the whole animal level.

[0058] 3) Proteomics-based pathway enrichment analysis of piglet jejunal tissue reveals the underlying mechanism by which suspensions antagonize DON toxicity. Based on the completion of the aforementioned animal experiments (Part 2) and the confirmation that the obtained suspension significantly improved the intestinal morphology of DON-infected piglets, this part of the experiment aims to utilize label-free quantitative proteomics technology to explore the molecular mechanism by which this composition alleviates DON intestinal toxicity from a systems biology perspective. Gene set enrichment analysis (GSEA) of key differentially expressed proteins will be performed to elucidate the potential targets of its protective effect at the pathway level.

[0059] The samples were directly derived from jejunal tissue collected from piglets at the end of Part 2 of the experiment. Jejunal tissue samples were randomly selected from six piglets each in the DON model group and Example 3 group, and flash-frozen in liquid nitrogen for proteomics analysis. Approximately 50 mg of jejunal tissue was weighed, added to lysis buffer, and thoroughly ground and sonicated to extract total protein. Protein concentration was determined using the BCA method. Equal amounts of protein were then subjected to reduction, alkylation, and trypsin digestion. The resulting peptides were labeled using a TMT 16-plex kit. Specifically, samples from six DON model groups were pooled into one pool, and samples from six triplet treatment groups were pooled into another pool. Technical reproducibility was performed to control for error. The labeled peptides were fractionated by high-performance liquid chromatography (HPLC) and then analyzed using a nano-flow HPLC system coupled with a high-resolution mass spectrometer (HMS).

[0060] The mass spectrometry acquisition mode was data-dependent. The raw mass spectrometry data was retrieved using Proteome Discoverer 2.4 software, with the UniProt database for pigs (Sus scrofa) as a reference. Strict screening criteria were set to identify and quantify proteins. Differentially expressed proteins (DEPs) were screened with a Fold Change (FC) ratio between the treatment group and the model group >1.2 or <0.833 and a P value <0.05 as the threshold. Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed on DEPs. Key steps included using GSEA 4.1.0 software, with all identified proteins as the background gene set (sorted by log2(FC)), to perform enrichment analysis on the "Hallmark" and "KEGG" gene sets to explore more subtle and systematic changes at the pathway level. Protein quantification data are expressed as mean ± standard deviation, and Student's test was used for intergroup comparisons. In GSEA analysis, a higher absolute value of the Normalized Enrichment Score (NES) indicates a stronger degree of enrichment, with positive or negative values ​​representing the trend of pathway activation (positive) or inhibition (negative). A nominal P-value < 0.05 was considered statistically significant.

[0061] The results are as follows Figure 3 As shown, the GSEA analysis results indicate that ( Figure 3 In Part A of the study, the apoptosis pathway showed a significant negative enrichment in the Example 3 group compared to the DON model group. The normalized enrichment score (NES) was -1.63, and the nominal P-value was 0.0077. The negative NES value indicates that a series of genes (proteins) related to the apoptosis pathway were generally downregulated in the ranking list. This means that the suspension treatment in Example 1 of this invention systematically inhibited the cell apoptosis-related protein network activated by DON challenge. This finding further demonstrates that the suspension obtained in this invention can downregulate the expression of pro-apoptotic genes (BAX, Caspase-3) and provides stronger evidence at the overall proteomic level. More importantly, GSEA analysis also revealed another significantly affected pathway—ferroptosis. Figure 3 (Part B). This pathway also showed significant negative enrichment, with an NES of -1.61 and a Nominal P-value of 0.0288. Ferroprepia is a novel iron-dependent, lipid peroxidation-driven programmed cell death pathway closely related to oxidative stress. Inhibition of this pathway suggests that the composition of this invention may block the DON-induced ferroptosis pathway by intervening in iron metabolism and lipid peroxidation processes. This finding provides a more upstream, pathway-level mechanistic explanation for the effect of the suspension obtained in Example 3 in significantly increasing the activity of antioxidant enzymes (SOD, CAT) and reducing the content of lipid peroxidation products (MDA). Inhibition of ferroptosis is a potential novel mechanism revealed in this invention, distinct from existing antidotes.

[0062] Proteomics and GSEA analysis of jejunal tissue elucidated the underlying mechanism by which the combination of hesperidin, sodium butyrate, and PR39 antimicrobial peptide alleviates DON-induced intestinal toxicity in piglets from a biological perspective. The composition provided by this invention systematically protects intestinal epithelial cells from DON toxicity damage through a multi-target, synergistic network of "antioxidant-antiferroptosis-antiapoptosis." The establishment of this integrated mechanism not only strengthens the scientific basis for its efficacy but also highlights its significant advancement and inventiveness compared to products with single mechanisms of action.

[0063] 4) Analysis of the gut microbiota in piglets' jejunal contents based on 16S rRNA gene sequencing reveals the regulatory effect of suspension on intestinal microecology. Based on the established fact that the suspension obtained in Example 3 of this invention can improve the growth performance and intestinal morphology of DON-infected piglets (Part 2), this part of the experiment aims to further explore its mechanism of action in alleviating DON toxicity from the perspective of intestinal microecological balance. High-throughput 16S rRNA gene sequencing technology was used to analyze the effects of the composition treatment on the jejunal flora structure of DON-infected piglets at the family, genus, and species levels, in order to elucidate its role in promoting intestinal health by regulating the intestinal flora.

[0064] The experimental samples were directly derived from Part 2). On day 28 of the experiment, at slaughter, jejunal contents were aseptically collected from piglets in the control group, DON model group, and Example 3 group. Six piglets were randomly selected from each group, resulting in a total of 12 samples. The samples were immediately placed in sterile cryovials, flash-frozen in liquid nitrogen, and then transferred to a -80°C ultra-low temperature freezer for storage and intestinal flora analysis. (1) Total microbial DNA was extracted from jejunal contents samples using a fecal genomic DNA extraction kit. The integrity and concentration of the DNA were detected by agarose gel electrophoresis and Nanodrop spectrophotometer. Using the extracted genomic DNA as a template, the V3-V4 variable region of the bacterial 16S rRNA gene was amplified by PCR using barcode-specific primers. After purification and quantification, the amplified products were sequenced by PE250 pairs on the Illumina NovaSeq 6000 platform. The raw data were subjected to quality control, including the removal of low-quality sequences and chimeras, to obtain effective sequences.

[0065] (2) Using QIIME2 (v2022.8) based on the Silva 138 database, sequences were clustered into operational taxonomic units (OTUs) with 97% similarity and species annotation was performed; data analysis and visualization were performed using packages such as ggplot2 and vegan in R language (v4.2.1); community richness and evenness were assessed by calculating the α diversity index; differences in community structure between groups were assessed by β diversity analysis; bar charts were used to display the community composition (relative abundance) of each group of samples at the family, genus, and species levels; differences in α diversity index between groups were analyzed using the Wilcoxon rank-sum test; differences in β diversity between groups were analyzed using PERMANOVA (Adonis test) based on the Bray-Curtis distance matrix; differences in relative abundance between groups of taxonomic units were screened using LEfSe analysis (LDA>2.0, P<0.05).

[0066] The results are as follows Figure 4 As shown, analysis of the jejunal contents microbiota revealed that, at different taxonomic levels, the treatment in Example 1 significantly regulated the DON-induced microbiota dysbiosis. Figure 4Part A): In the DON model group, the relative abundance of bacterial families associated with inflammation and intestinal barrier damage (such as Clostridiaceae) was significantly increased compared to the control group (CON). However, after treatment in Example 1, the relative abundance of beneficial bacterial families Lactobacillaceae and Oscillospiraceae was significantly restored or increased, while the abundance of Clostridiaceae decreased to levels close to the control group. Genus level ( Figure 4 Part B: DON challenge led to an increase in the abundance of opportunistic pathogens and an inhibition of the abundance of beneficial bacteria, Lactobacillus. However, the treatment in Example 3 of this invention effectively reversed this trend, significantly increasing the relative abundance of Lactobacillus while reducing the proportion of some harmful Clostridium species. Species level ( Figure 4 (Part C): Analysis showed that DON exposure altered the abundance of several specific bacterial species. The treatment group in Example 4 significantly promoted the colonization of lactobacillus species known to have probiotic functions (such as Lactobacillus reuteri or Lactobacillus samylovorus) and inhibited the proliferation of specific harmful bacterial species associated with increased DON toxicity.

[0067] In summary, DON challenge disrupted the homeostasis of the jejunal intestinal flora in piglets, resulting in a decrease in beneficial bacteria, an increase in potentially harmful bacteria, and a decline in flora diversity. However, the suspension obtained in Example 4 of this invention can effectively reshape the intestinal flora structure. Its effects include: significantly promoting the proliferation of beneficial bacteria; inhibiting the overgrowth of opportunistic pathogens; and promoting the overall flora structure to revert to that of the healthy control group.

[0068] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A suspension for improving DON-induced enteric toxicity in piglets, characterized in that, Including the following raw materials: Hesperidin 40-60 mg / mL, sodium butyrate 50-150 mg / mL and PR-39 antimicrobial peptide 10-15 mg / mL.

2. The method for preparing a suspension for improving DON-induced enteric toxicity in piglets as described in claim 1, characterized in that, Includes the following steps: Sodium butyrate and hesperidin were homogenized under high pressure in a pH buffer matrix, and then PR-39 antimicrobial peptide was added under stirring. After adjusting the pH, the suspension was obtained.

3. The method for preparing a suspension for improving DON-induced enteric toxicity in piglets according to claim 1, characterized in that, The pH buffer matrix includes 0.5% hydroxypropyl methylcellulose and 0.1% Tween-80.

4. The method for preparing a suspension for improving DON-induced enteric toxicity in piglets according to claim 1, characterized in that, The solvent for the pH buffer matrix is ​​water.

5. The method for preparing a suspension for improving DON-induced enteric toxicity in piglets according to claim 1, characterized in that, The pH adjustment is to adjust the pH to 7.

0.

6. The method for preparing a suspension for improving DON-induced enteric toxicity in piglets according to claim 1, characterized in that, The pressure of the high-pressure homogenizer is 80 MPa, and the temperature is 25℃.

7. The method for preparing a suspension for improving DON-induced enteric toxicity in piglets according to claim 1, characterized in that, The stirring temperature is 25°C and the stirring speed is 300 rpm.

8. The use of the suspension as described in claim 1 in the preparation of a drug for improving DON-induced enteric toxicity in piglets.

Images