Use of a composition in the manufacture of a product for alleviating salmonella infection

Abstract

The application discloses application of a composition in preparation of a product for relieving salmonella infection, and relates to the technical field of biotechnology.The composition comprises the following components in parts by weight: ganoderma lucidum polysaccharide 50-70 parts, jerusalem artichoke polysaccharide 25-35 parts, lentinan 25-35 parts, polygonatum officinale powder 15-25 parts and biota leaf 15-35 parts.The composition provided by the application can regulate immune inflammatory balance, repair intestinal structure damage, target-regulate intestinal flora structure and metabolic function, reshape cecal flora structure, enrich anti-inflammatory beneficial bacteria, inhibit pathogenic bacteria, enhance the carbon metabolism capacity of the flora, and effectively relieve the negative damage caused by salmonella infection to laying hens.The application provides a theoretical basis and data support for the composition as a green feed additive for preventing and controlling poultry salmonella disease.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the use of a composition in the preparation of products that alleviate Salmonella infection. Background Technology

[0002] Salmonella is one of the most serious foodborne pathogens in large-scale poultry farming. It has a wide spread and high infection rate in laying hen farming, spreading vertically from breeder chickens to chicks, and horizontally through feed, water, cages, and human contact, easily causing outbreaks in flocks. When laying hens are infected with Salmonella, it first damages the intestinal mucosa, disrupting the normal physiological structure and barrier function of the intestines, and interfering with the body's immune homeostasis. This leads to problems such as lethargy, decreased feed intake, reduced egg production, and lighter egg weight, significantly lowering poultry production performance. Simultaneously, the pathogen can invade the egg, causing egg contamination. Contaminated eggs and chicken meat entering the market become a significant source of Salmonella food poisoning in humans, seriously threatening public health and food safety, and causing significant economic losses to the poultry farming and food industries.

[0003] For a long time, farms have relied heavily on antibiotics to control Salmonella infections. While these drugs can inhibit the proliferation of pathogens and alleviate symptoms in the short term, the drawbacks of long-term, frequent, and even indiscriminate use of antibiotics are becoming increasingly apparent. On the one hand, the continuous pressure of drug selection will cause Salmonella to gradually develop multidrug resistance, forming drug-resistant strains, which will significantly reduce the effectiveness of subsequent conventional drug treatments, and may even lead to a situation where no drugs are available. On the other hand, antibiotics cannot be completely metabolized by the body, easily forming veterinary drug residues in poultry products such as eggs and chicken. Long-term ingestion of these residues by humans can damage organs such as the liver and kidneys, and can also induce intestinal flora imbalance and the development of drug-resistant bacteria, creating hidden health risks. In addition, while antibiotics kill harmful bacteria, they also indiscriminately destroy beneficial microorganisms in the intestines of laying hens, further exacerbating the imbalance of the intestinal microecology, reducing the flock's own resistance, and forming a vicious cycle of "medication - decreased physical condition - repeated infection - increased dosage".

[0004] With the standardization of the livestock farming industry, antibiotic-free farming has become a mandatory requirement and mainstream trend for the transformation and upgrading of the poultry industry and for ensuring food safety. Against this industry backdrop, developing new green disease control products that are naturally derived, safe, non-toxic, residue-free, and unlikely to induce drug resistance has become a research hotspot in the field of livestock and poultry disease prevention and control. Summary of the Invention

[0005] The purpose of this invention is to provide an application of a composition in the preparation of products that alleviate Salmonella infection, thereby addressing the problems existing in the prior art. The composition provided by this invention can effectively alleviate the negative damage caused by Salmonella infection to laying hens by regulating immune inflammation balance, repairing intestinal structural damage, and targeting and regulating the structure and metabolic function of the intestinal flora.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides the application of a composition in the preparation of a product for alleviating Salmonella infection, the composition comprising the following components in parts by weight: 50-70 parts of Ganoderma lucidum polysaccharide, 25-35 parts of Jerusalem artichoke polysaccharide, 25-35 parts of Lentinus edodes polysaccharide, 15-25 parts of Polygonatum odoratum powder, and 15-35 parts of Platycladus orientalis leaf.

[0007] Preferably, the composition comprises the following components in parts by weight: 60 parts of Ganoderma lucidum polysaccharide, 30 parts of Jerusalem artichoke polysaccharide, 30 parts of shiitake mushroom polysaccharide, 20 parts of Solomon's seal powder, and 20 parts of Platycladus orientalis leaf.

[0008] Furthermore, the product is a feed additive.

[0009] Furthermore, the feed additive is a layer hen feed additive.

[0010] Furthermore, the product is a veterinary drug.

[0011] The present invention also provides a product for alleviating Salmonella infection, wherein the active ingredient comprises the above-described composition.

[0012] Furthermore, the product is a feed additive.

[0013] Furthermore, the feed additive is a layer hen feed additive.

[0014] Furthermore, the product is a veterinary drug.

[0015] The present invention discloses the following technical effects: The composition provided by this invention can effectively alleviate the negative damage caused by Salmonella infection to laying hens by regulating the balance of immune inflammation, repairing intestinal structural damage, and targeting and regulating the structure and metabolic function of intestinal flora. This provides a theoretical basis and data support for the use of this composition as a green feed additive to prevent and control poultry salmonellosis.

[0016] Immune organ indices are core indicators for evaluating the innate immune function of poultry, and intestinal weight index directly reflects intestinal development and nutrient absorption capacity. This invention found that Salmonella infection significantly increased the spleen and liver indices in laying hens, and significantly decreased the weight indices of the duodenum, ileum, and cecum, suggesting that Salmonella invasion induces stress-induced swelling of immune organs while inhibiting normal intestinal development, resulting in impaired intestinal nutrient absorption. Under infection conditions, this composition can significantly reverse the abnormal swelling of immune organs and shows a recovery trend in the damaged intestinal weight index. Furthermore, the composition has a significant main effect on the liver and cecum indices, indicating that it can alleviate Salmonella-induced immune stress, improve intestinal development, and enhance the body's stress resistance by regulating organ development homeostasis.

[0017] Serum pro-inflammatory cytokine levels are a direct reflection of the body's systemic inflammatory response. Excessive secretion of pro-inflammatory factors such as IL-1β, IFN-γ, and TNF-α can exacerbate tissue inflammatory damage. Adding the composition of this invention to the diet can significantly reduce serum IFN-γ levels in both healthy and infected laying hens, and shows a downregulation trend for pro-inflammatory factors such as IL-1β and IL-6. Furthermore, this composition exhibits a significant negative main effect on IFN-γ, indicating that it possesses broad-spectrum anti-inflammatory activity. It can alleviate Salmonella-induced systemic inflammatory damage by inhibiting the excessive release of pro-inflammatory cytokines, maintain the body's immune inflammatory balance, and reduce secondary damage to body tissues caused by the inflammatory response.

[0018] The gut is the largest immune organ in poultry. The TLRs / NF-κB and MAVS-mediated IFN-α / β and MAPK signaling pathways are core signaling pathways regulating intestinal mucosal immunity and resistance to bacterial infection. This invention shows that Salmonella infection significantly activates the jejunal TLR2 / NF-κB pathway and upregulates the expression of pro-inflammatory factors and antiviral-related IFN-α / β genes, suggesting that Salmonella activates downstream inflammatory and immune signals by recognizing pattern receptors, inducing an excessive immune response in the intestinal mucosa. The composition of this invention significantly downregulates the expression of TLR2, NF-κB, IL-1β, IL-17, and IFN-α / β genes in the gut of infected laying hens, while showing an upregulation trend for anti-inflammatory genes. Furthermore, there is a significant interaction between these factors on key genes such as TLR2, NF-κB, and NOD2. These results indicate that this composition can inhibit... TLRs / NF-κB Inflammatory pathways are activated and the MAVS / IFN pathway homeostasis is regulated, which can alleviate Salmonella-induced excessive inflammation of the intestinal mucosa, enhance the intestinal mucosal immune defense capacity, and maintain the stability of the intestinal immune microenvironment.

[0019] Intestinal villus height, crypt depth, and V / C ratio are key indicators for evaluating intestinal mucosal barrier integrity and nutrient absorption. Villus atrophy and crypt hyperplasia directly impair intestinal absorption and physical barriers. This invention demonstrates that Salmonella infection significantly reduces jejunal villus height and V / C ratio, and significantly increases crypt depth, causing typical intestinal mucosal structural damage. This is consistent with research findings on Salmonella's disruption of intestinal epithelial cell integrity. Adding the composition of this invention to the diet not only significantly improves intestinal villus development in healthy laying hens but also effectively reverses infection-induced intestinal morphological damage, significantly restores villus height and V / C ratio, and reduces crypt depth. Furthermore, there is a significant interaction between these two factors regarding villus height and crypt depth. This indicates that the composition can directly promote intestinal epithelial cell proliferation and development, repair physical damage to the intestinal mucosa caused by Salmonella, maintain intestinal structural integrity, and provide a structural basis for nutrient absorption and barrier defense.

[0020] Intestinal tight junction proteins (ZO-1, occludin, claudin-1) are core components of the intestinal chemical barrier. The expression levels of apoptosis genes Bcl2 and Caspase9, and the antioxidant gene Nrf2 are directly related to the survival and antioxidant capacity of intestinal epithelial cells. GPR41 / 43 are key receptors mediated by short-chain fatty acids in intestinal health. This invention found that Salmonella infection significantly downregulated the expression of tight junction proteins, the anti-apoptotic gene Bcl2, the antioxidant gene Nrf2, and GPR receptor genes, and significantly upregulated the expression of the pro-apoptotic gene Caspase9, suggesting that Salmonella disrupts the intestinal tight junction barrier, induces epithelial cell apoptosis, and inhibits antioxidant and metabolic signaling pathways. The composition of this invention can significantly downregulate Caspase9 expression in the intestine of infected laying hens, upregulate GPR43 gene levels, and show an upregulatory trend for tight junction proteins and anti-apoptotic genes, with a significant interaction between the two on Caspase9. The results, combined with those of short-chain fatty acids, indicate that this composition can enhance the integrity of intestinal tight junctions, inhibit epithelial cell apoptosis, and activate the GPR receptor signaling pathway, thereby constructing a complete intestinal barrier defense system and blocking the intestinal invasion and translocation of Salmonella.

[0021] The gut microbiota is a core regulator of intestinal health in poultry, and its diversity, structure, and metabolic function are directly related to the body's ability to resist pathogen infection. In this invention, Salmonella infection significantly reduces cecal microbiota α-diversity, disrupts microbiota structure, enriches pathogenic bacteria such as Helicobacter spp., and reduces the abundance of beneficial symbiotic bacteria, confirming that Salmonella can disrupt intestinal microecological homeostasis and provide a favorable environment for pathogen colonization. The composition of this invention can reshape the gut microbiota structure, showing a recovery trend from infection-induced reduction in microbiota diversity, and its regulation of key bacterial genera is infection-state dependent: in a healthy state, the composition significantly enriches beneficial bacteria with polysaccharide metabolism, such as Bacteroides spp.; in an infected state, it directionally increases the abundance of anti-inflammatory beneficial bacteria (RC9 from the RIKEN family) and inhibits the proliferation of pathogenic bacteria such as Desulfovibrio spp. Tax4Fun functional prediction further confirms that the composition can enhance the activity of microbiota carbohydrate and energy metabolism pathways, providing energy for intestinal metabolism. The above results indicate that the composition reverses Salmonella-induced dysbiosis and constructs an anti-colonization gut microbiota barrier by optimizing microbial diversity, targeting and regulating the abundance of core functional bacteria, and enhancing microbial metabolic function.

[0022] Intestinal metabolites are key mediators of microbial-host interactions, directly reflecting intestinal microecological metabolic activity and the host's nutritional metabolic status. PCA and P LS-DA analysis showed that the composition of this invention significantly altered the overall metabolic profile of the cecum in healthy laying hens, identifying 160 differentially expressed metabolites, with core regulation of microbial-mediated pathways such as sphingolipid metabolism, amino acid metabolism, and bile secretion. The composition significantly upregulated anti-inflammatory and barrier-protective metabolites such as prostaglandin derivatives and neuraminic acid, while downregulating metabolites such as glycosides and flavonoids. This suggests that the composition can improve nutrient utilization efficiency by regulating intestinal lipid and amino acid metabolism, while simultaneously enhancing intestinal anti-inflammatory and mucosal protective capabilities. It optimizes the intestinal microenvironment at the metabolic level, providing metabolic support for overall health. This complements the results of microbiota structure regulation, confirming the synergistic regulatory effect of the composition on the intestinal microecology of microbiota and metabolites.

[0023] Short-chain fatty acids (SCFAs) are core metabolites of polysaccharides fermented by gut microbiota, possessing multiple functions including energy supply, anti-inflammation, and GPR receptor activation. They serve as a crucial bridge connecting the gut microbiota and host health. Test results from healthy laying hens show that the composition of this invention significantly increases the concentrations of butyric acid and valerate in the cecum, and exhibits an increasing trend in propionic acid and isohexanoic acid. This is highly consistent with the results of Bacteroides enrichment and enhanced carbohydrate metabolism pathways observed in gut microbiota sequencing. Combined with gut gene expression results, it is evident that this composition achieves a cascade regulation of the intestinal barrier and immune inflammation through the signaling axis of enriching beneficial polysaccharide-metabolizing bacteria → promoting SCFA synthesis → activating jejunal GPR41 / 43 receptors. Butyric acid, as the core functional SCFA, provides energy to intestinal epithelial cells, strengthens tight junctions, and inhibits inflammatory responses. It is the core metabolic mediator for the composition's intestinal protective effect, thus constructing a complete regulatory closed loop of composition-gut microbiota-SCFA-intestinal health. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 Principal coordinate analysis (PCoA) plot showing the effect of dietary supplementation with a combination of ingredients on the cecal microbial community structure of laying hens infected with Salmonella; Figure 2 Bar chart showing the relative abundance of the top 10 microorganisms in the cecum genus of Salmonella-infected laying hens when dietary supplementation composition was added. Figure 3 Independent samples t-test plot of differentially expressed bacteria at the cecal level in laying hens between the blank control group and the challenge group; Figure 4 Bar graph of independent samples t-test for differential bacterial levels in the cecum of laying hens between the blank control group and the composition group; Figure 5 Bar plot of independent samples t-test for differential bacterial levels in the cecum of laying hens between the challenge group and the challenge + combination group; Figure 6 LEfSe analysis of LDA effect size bar graph for differential biomarkers of cecal microbiota in laying hens under different treatment groups; Figure 7 Abundance distribution of KEGG functional pathway (Level 2) in the cecal microbiota of laying hens in different treatment groups; Figure 8 Principal component analysis (PCA) diagram of non-targeted metabolomics of cecal contents in laying hens; Figure 9Partial least squares discriminant analysis (PLS-DA) plot of non-targeted metabolomics of cecal contents in laying hens in the blank control group and the composition group; Figure 10 Volcano plot of differential metabolites in the cecal non-targeted metabolite group of laying hens between the blank control group and the composition group; Figure 11 This is a graph showing the enrichment analysis of differential metabolites in the cecum of laying hens between the blank control group and the composition group, specifically the KEGG pathway. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0029] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0030] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0031] Terminology Explanation: Yu Zhu, a Chinese medicinal herb, is the rhizome of Polygonatum odoratum (Mill.) Druce, a plant belonging to the genus Polygonatum in the family Liliaceae.

[0032] Platycladus orientalis (L.) franco, a Chinese medicinal herb, refers to the branches and leaves of Platycladus orientalis (L.) franco, a plant belonging to the genus Platycladus in the family Cupressaceae.

[0033] The Ganoderma lucidum polysaccharide used in the following examples was purchased from Shaanxi Senfu Natural Products Co., Ltd.; Jerusalem artichoke polysaccharide was purchased from Shandong Yidelai Biotechnology Co., Ltd.; and Lentinan was purchased from Shandong Xinde Technology Co., Ltd.

[0034] Example 1 A method for preparing a composition for alleviating Salmonella infection: (1) After crushing the Solomon's Seal and the leaves of Platycladus orientalis into powder, pass them through a 60-mesh sieve to obtain Solomon's Seal powder and Platycladus orientalis leaf powder.

[0035] (2) Mix 60g of Ganoderma lucidum polysaccharide, 30g of Jerusalem artichoke polysaccharide, 30g of shiitake mushroom polysaccharide, 20g of Solomon's seal powder and 20g of Platycladus orientalis leaf powder evenly to obtain the composition.

[0036] Example 2 A method for preparing a composition for alleviating Salmonella infection: (1) After crushing the Solomon's Seal and the leaves of Platycladus orientalis into powder, pass them through a 60-mesh sieve to obtain Solomon's Seal powder and Platycladus orientalis leaf powder.

[0037] (2) Mix 50g of Ganoderma lucidum polysaccharide, 35g of Jerusalem artichoke polysaccharide, 25g of shiitake mushroom polysaccharide, 15g of Solomon's seal powder and 35g of Platycladus orientalis leaf powder evenly to obtain the composition.

[0038] Example 3 A method for preparing a composition for alleviating Salmonella infection: (1) After crushing the Solomon's Seal and the leaves of Platycladus orientalis into powder, pass them through a 60-mesh sieve to obtain Solomon's Seal powder and Platycladus orientalis leaf powder.

[0039] (2) Mix 70g of Ganoderma lucidum polysaccharide, 25g of Jerusalem artichoke polysaccharide, 35g of shiitake mushroom polysaccharide, 25g of Solomon's seal powder and 15g of Platycladus orientalis leaf powder evenly to obtain the composition.

[0040] Example 1 of effect verification 1. Materials and Methods 1.1 Test Materials The composition prepared in Example 1; Salmonella Typhimurium (CVCC 2232) was purchased from the National Veterinary Microbiology Culture Collection Center; the serum cytokine ELISA kit (Shanghai Enzyme-Link Biotechnology Co., Ltd.), total RNA extraction kit, reverse transcription kit, and real-time quantitative PCR (qPCR) reagent (Takara Biotech (Beijing) Co., Ltd.) were all commercially available analytical grade products; 16S rRNA sequencing, non-targeted metabolomics, and short-chain fatty acid detection were all performed by a professional biotechnology company; primers were designed and synthesized based on GenBank sequences.

[0041] 1.2 Experimental Animals and Experimental Design The experiment used healthy Hy-Line Brown chickens aged 81 weeks and adopted a 2×2 two-factor completely randomized experimental design. The main effects included Salmonella challenge (- / +) and composition addition (0 / 600 mg / kg). There were 4 treatment groups and 6 replicates in each group.

[0042] The experimental groups are as follows: NC Group: Basic daily rations, no poison attacks; GLP group: basal diet + 600 mg / kg combination, no challenge; PC group: Basic diet, Salmonella challenge; SGLP group: basal diet + 600 mg / kg combination, challenged with Salmonella.

[0043] Chickens in the above experimental groups were fed a basal diet or a basal diet supplemented with the above-mentioned composition for 12 weeks. Seven days before the end of the experiment, the challenge group was challenged with Salmonella. The challenge procedure was as follows: Once, on the day of challenge, chickens in the PC and SGLP groups were orally administered 1 mL of 1 × 10⁻⁶ Salmonella. 9 CFU / mL Salmonella Typhimurium culture medium. On the same day, the NC and GLP groups were inoculated with 1.0 mL of blank liquid culture medium (Beijing Luqiao Technology Co., Ltd., CM201) that did not contain Salmonella Typhimurium.

[0044] 1.3 Feeding and Management The basal diet was formulated according to the "NY / T33-2021 Chicken Feeding Standard" as a corn-soybean meal complete diet. The diet was in powder form, and the formula and nutrient levels are shown in the appendix to the main text. The experimental chickens were housed in two-tiered cages, with three chickens per cage, and were allowed free access to feed and water. Standardized feeding management was implemented throughout the process. The temperature in the chicken house was controlled at 22-26℃, the relative humidity at 55-65%, and the lighting regime was 16 hours of light: 8 hours of darkness, with a light intensity of 15-20 lx. Manure was removed and ventilation was carried out regularly every day to keep the house clean and dry. No antibiotics, probiotics, or other feed additives were used during the experiment to prevent external factors from interfering with the experimental results. The formula and nutrient levels of the laying hen diet are shown in Table 1.

[0045] Table 1. Dietary formulation and nutrient levels (%, air-dried basis) Note: ª Premix provides vitamins, trace elements, and carriers per kilogram of ration (formulated according to laying hen feeding standards); ᵇ All nutrient levels are calculated values.

[0046] 1.4 Sample Collection At the end of the experiment, 7 days after the challenge, one chicken was randomly selected from each replicate. Blood was collected from the jugular vein to separate serum, which was stored at -20°C for later use. After slaughter, the spleen, liver, and intestinal segments were quickly separated, and the organ indices were calculated. The middle section of the jejunum was cut to prepare tissue sections. The cecal mucosa and contents were scraped, flash-frozen in liquid nitrogen, and stored at -80°C for microbial sequencing, metabolomics, and short-chain fatty acid detection. Jejunal tissue was used for gene expression assays.

[0047] 1.5 Measurement Indicators and Methods 1.5.1 Weight index of immune organs and intestines Weigh the spleen, liver, duodenum, jejunum, ileum, and cecum, and calculate the organ index and intestinal weight index using the following formula: Organ index (%) = organ weight (g) / live body weight (g) × 100%.

[0048] 1.5.2 Serum cytokine levels The levels of IL-1β, IFN-γ, IL-6, and TNF-α in serum were measured using an ELISA kit, and the procedure was strictly followed according to the kit instructions.

[0049] 1.5.3 Jejunal tissue morphology and structure After fixation, dehydration, embedding, sectioning, and HE staining, the villus height and crypt depth were measured under a microscope, and the villus-crypt ratio (V / C) was calculated.

[0050] 1.5.4 Jejunum gene mRNA expression level Total RNA was extracted using the Trizol method, and cDNA was synthesized by reverse transcription. qPCR was used to determine the expression levels of genes related to the intestinal barrier (ZO-1, occludin, claudin-1), apoptosis (Bcl2, Caspase9), antioxidant (Nrf2), short-chain fatty acid receptors (GPR41, GPR43), and TLRs / NF-κB and MAVS / IFN-α / β immune pathways. GA... P DH is an internal parameter, using 2 -ΔΔCt The relative expression level is calculated using this method.

[0051] 1.5.5 High-throughput sequencing of cecal microorganisms' 16S rRNA Total microbial DNA was extracted from the contents of the cecum, and the 16S rRNA V3-V4 region was amplified and sequenced using the Illumina platform. α-diversity, β-diversity, species composition, differentially expressed bacteria between groups (t-test), and Tax4Fun functional prediction analyses were performed.

[0052] 1.5.6 Non-targeted metabolomics analysis of cecal contents Non-targeted metabolic profiling was performed using LC-MS / MS, and PCA was used to detect the metabolites. P LS-DA was used to screen for differentially expressed metabolites and to perform KEGG pathway enrichment analysis.

[0053] 1.5.7 Cecal short-chain fatty acid content The concentrations of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, hexanoic acid, and isohexanoic acid were determined by gas chromatography, with units of μg / mg.

[0054] 1.6 Data Statistical Analysis The main effects and interactions between Salmonella challenge and composition addition were analyzed using a two-way ANOVA in a general linear model using SPSS 26.0 (SPSS Inc., USA) software. If an interaction effect was found, one-way ANOVA and Duncan's multiple comparisons were performed. P <0.05 indicates a significant difference; 0.05 < P <0.10 indicates a divergent trend.

[0055] 2 Results 2.1 Organ Index As shown in Table 2, compared with the control group, Salmonella infection significantly increased the spleen index and liver index in laying hens. P <0.001), but significantly reduced the weight index of the duodenum, ileum, and cecum ( P <0.05), showing a decreasing trend in jejunal weight index (0.05 < P <0.10). Under uninfected conditions, the addition of the composition tended to reduce liver indices (0.05 < 0.10). P <0.10), with a decreasing trend in the weight index of the duodenum, ileum, and cecum (0.05 < P <0.10). Under Salmonella infection conditions, the addition of the composition significantly reduced the elevated spleen and liver indices caused by infection ( P <0.05), and showed a recovery trend in the reduced weight index of the duodenum and cecum (0.05 < P <0.10). Salmonella infection had a significant main effect on all organ index indicators ( P <0.05%, the addition of the composition had a significant main effect on liver index and cecal weight index ( ). P<0.05), but there was no significant interaction between the two ( P >0.05).

[0056] Table 2. Effects of dietary supplementation on immune organ index and intestinal weight index in laying hens infected with Salmonella. Note: Different lowercase letters in the superscript of data in the same column indicate significant differences. P <0.05), the presence of both different and identical letters on the shoulder insignia indicates a trend of discrepancy (0.05 < P <0.10, with the same letter or no letter on the shoulder label indicating no significant difference ( P >0.05). Same as the table below.

[0057] 2.2 Serum cytokines As shown in Table 3, compared with the control group, Salmonella infection significantly increased the serum levels of IL-1β, IFN-γ, IL-6, and TNF-α. P <0.05%. Under uninfected conditions, the addition of the composition significantly reduced IFN-γ levels ( P <0.05), showing a trend of decreasing levels of IL-1β and TNF-α (0.05 < P <0.10). Under Salmonella infection conditions, the addition of the composition significantly reduced serum IFN-γ levels ( P <0.05), showing a decreasing trend towards IL-1β and IL-6 (0.05 < P <0.10). Salmonella infection had a significant main effect on all cytokines ( P <0.05%, the addition of the composition had a significant main effect of reducing IFN-γ ( P <0.05), showing a trend towards reducing the main effects on IL-1β and IL-6 (0.05 < P <0.10, with no significant interaction between the two ( P >0.05).

[0058] Table 3. Effects of dietary supplementation composition on serum cytokine levels in laying hens infected with Salmonella. 2.3 Jejunal tissue morphology and structure As shown in Table 4, compared with the control group, Salmonella infection significantly reduced the villus height and V / C ratio in the jejunum of laying hens. P <0.05, significantly increasing crypt depth ( P <0.001). Under uninfected conditions, dietary supplementation with the composition at 600 mg / kg significantly increased villus height, crypt depth, and V / C ratio ( ). P<0.05%. Under Salmonella infection conditions, the addition of the composition significantly alleviated infection-induced intestinal morphological damage, manifested as a significant increase in villous height and V / C ratio reduced due to infection ( P <0.05, and significantly reversed the abnormally increased crypt depth caused by infection ( P <0.05). Salmonella infection had a significant main effect on all three indicators mentioned above ( P <0.05%, the addition of the composition has a significant main effect on the pile height and V / C ratio ( P <0.05). There is a significant interaction between the two in terms of villus height and crypt depth ( P <0.05).

[0059] Table 4. Effects of dietary additives on the morphology and structure of jejunal tissue in laying hens infected with Salmonella. 2.4 Jejunal intestinal barrier function, apoptosis, antioxidant activity, and expression of short-chain fatty acid receptor-related genes Table 5 shows that, compared with the control group, Salmonella infection in laying hens significantly reduced the mRNA levels of ZO1, claudin-1, occludin, Bcl2, Nrf2, GPR41, and GPR43 in the jejunum. P <0.05), and significantly increased the mRNA level of Caspase9 ( P <0.05%. Under uninfected conditions, dietary supplementation with the composition significantly increased the mRNA levels of Nrf2, GPR41, and GPR43 in the jejunum of laying hens ( P <0.05, significantly reduced Caspase9 mRNA levels ( P <0.05), showing a trend of increasing mRNA levels of ZO-1, claudin-1, and occludin (0.05 < P <0.10). Under Salmonella infection conditions, the addition of the composition significantly reduced the mRNA level of Caspase 9 ( P <0.05, significantly increased GPR43 mRNA levels ( P <0.05), showing a trend of increasing mRNA levels of claudin-1, occludin, and Bcl2 (0.05 < P <0.10). The two only showed a significant interaction on Caspase9 ( P = 0.010).

[0060] Table 5. Effects of dietary supplementation combinations on jejunal intestinal barrier function, apoptosis, antioxidant activity, and expression of short-chain fatty acid receptor-related genes in Salmonella-infected laying hens. 2.5 Jejunal immune function and expression of genes related to the TLRs / NF-κB signaling pathway As shown in Tables 6 and 7, compared with the control group, Salmonella infection in laying hens significantly increased jejunal iNOS and IgA levels. P mRNA levels of IgR, TLR2, NF-κB, IL-1β, IL-4, IFN-γ, TNF-α, IL-6, IL-10, and IL-17 ( P <0.05), showing a trend of increasing TLR4 mRNA levels (0.05< P <0.10). Under uninfected conditions, dietary supplementation significantly increased the mRNA levels of pIgR and TLR2 in the jejunum of laying hens ( P <0.05), showing a trend of increasing mRNA levels of iNOS, IgA, TLR4, NF-κB, IL-4, and IL-10 (0.05 < P <0.10). Under Salmonella infection conditions, the addition of the composition significantly reduced the mRNA levels of TLR2, NF-κB, IL-1β, and IL-17 in the jejunum ( P <0.05), showing a decreasing trend in the mRNA levels of iNOS, TLR4, IFN-γ, and TNF-α (0.05 < P <0.10), showing a trend of increasing IL-4 and IL-10 mRNA levels (0.05< P <0.10). Salmonella infection has a significant main effect on the above genes ( P <0.05, and the two have significant interactions on TLR2, NF-κB, IL-1β and IL-17 ( P <0.05).

[0061] Table 6. Effects of dietary supplementation combinations on jejunal immune function and expression of genes related to the TLRs / NF-κB signaling pathway in Salmonella-infected laying hens. Table 7 Effects of dietary supplementation composition on cytokines 2.6 Expression of genes related to jejunal MAVS and IFN-α / β signaling pathways As shown in Table 8, compared with the control group, Salmonella infection in laying hens significantly increased the mRNA levels of NOD2, TRAF6, IRF3, IFN-α, and IFN-β in the jejunum. P <0.05), for NOD5, P The mRNA level at 38 showed an increasing trend (0.05 < P<0.10). Under uninfected conditions, dietary supplementation with the composition significantly increased the mRNA levels of NOD2, JUK, and TRAF6 in the jejunum of laying hens ( P <0.05), for NOD1, NOD5, TAK1, P 38. The mRNA level of IFN-α showed an increasing trend (0.05 < P <0.10), showing a decreasing trend in IKKε mRNA levels (0.05< P <0.10). Under Salmonella infection conditions, the addition of the composition significantly reduced the mRNA levels of jejunal IKKε, IFN-α, and IFN-β ( P <0.05), for NOD1, NOD2, NOD5, TAK1 and P The mRNA level at 38 showed a decreasing trend (0.05 < P <0.10). Salmonella infection had a significant main effect on the expression of NOD2, TRAF6, IRF3, IFN-α, and IFN-β. P <0.05%. The addition of the composition had a significant main effect on the expression of JUK and IKKε ( P <0.05. Significant interactions exist between the two on NOD2, TRAF6, IFN-α, and IFN-β. P <0.05).

[0062] Table 8 Effects of dietary supplementation composition on gene expression related to the MAVS-mediated IFN-α / β and MAPK signaling pathway in the jejunum of Salmonella-infected laying hens 2.7 Cecal Microbiota 2.7.1 Alpha Diversity As shown in Table 9, compared with the control group, Salmonella infection significantly reduced the Chao1 and Shannon indices of cecal microorganisms in laying hens. P <0.05. Under uninfected conditions, dietary supplementation with 600 mg / kg of the composition had no significant effect on either the Chao1 index or the Shannon index. P >0.05). Under Salmonella infection conditions, the addition of the composition tended to increase the Chao1 and Shannon indices (<0.05). P <0.10). Salmonella infection had a significant main effect on the expression of the Chao1 index ( P <0.05. The addition of the composition and its interaction had no significant effect on either diversity index ( P >0.05).

[0063] Table 9. Effects of dietary supplementation combinations on α-diversity of cecal microorganisms in Salmonella-infected laying hens. 2.7.2 β-diversity The effects of dietary supplementation combinations on the cecal microbiota structure of Salmonella-infected laying hens were investigated using PCoA analysis based on Bray-Curtis distance combined with the ANOSIM test. Figure 1 PCoA results showed that PC1 and PC2 together explained 46.24% of the gut microbiota variation. The NC and GLP groups were completely separated with good intragroup aggregation; the PC and SGLP groups were intermediate, showing a trend towards regressing to healthy gut microbiota. ANOSIM test indicated that the composition of this invention can significantly remodel the cecal microbiota structure of healthy laying hens. P <0.05, and effectively reversed the bacterial community shift caused by Salmonella challenge ( P <0.05), maintaining gut microbiota homeostasis.

[0064] 2.7.3 Top 10 microorganisms at the genus level The top 10 bacterial groups at the Salmonella genus level in laying hens affected by dietary supplementation are shown in the table. Figure 2 The effects of Salmonella infection on the relative abundance of key bacteria in the Top 10 cecal microorganisms are shown in Table 10. Two-way ANOVA showed that Salmonella infection and the addition of the composition had a significant impact on the abundance of Bacteroides (…). Bacteroides ), Riken Bacteria Family RC9 Intestinal Group ( Rikenellaceae_RC9_gut_group The relative abundance of ) has a significant interaction effect ( P <0.05: Before challenge, the addition of the composition significantly increased the abundance of Bacteroides; after challenge, the composition significantly decreased its abundance. P <0.05%, restoring it to the NC group level. In the absence of the composition, Salmonella challenge significantly reduced the abundance of the Riken Bacteriaceae RC9 intestinal flora ( P <0.05), while the composition could completely reverse this effect, resulting in a significantly higher abundance in the SGLP group than in the PC group ( P <0.05. The abundance of *Desulfovibrio* in the PC group was significantly higher than that in the NC group. P <0.05); the added composition showed a tendency to increase the activity of *Desulfovibrio* spp. under uninfected conditions (0.05 < 0.05). P <0.10), but it showed a decreasing trend under infected conditions (0.05< P <0.10). Lactobacillus ( Lactobacillus The relative abundance did not differ significantly among the groups. P >0.05).

[0065] The above results indicate that the regulatory effect of the composition of the present invention on key cecal bacteria is highly dependent on the host infection state: in a healthy state, it promotes the proliferation of Bacteroides, a polysaccharide-metabolizing beneficial bacterium; in an infected state, it significantly increases the abundance of the anti-inflammatory probiotic Riken Bacteria RC9 intestinal flora and inhibits the excessive growth of the conditionally pathogenic Desulfovibrio, demonstrating its potential to reshape the intestinal flora structure under pathological conditions.

[0066] Table 10 Effects of dietary supplementation compositions on the relative abundance of key bacteria in the top 10 microorganisms in the cecal spp. of Salmonella-infected laying hens. 2.7.4 T-test for differentially expressed microorganisms Depend on Figure 3 As can be seen, the independent samples t-test results show that, compared with the NC group, the PC group had a lower incidence of Helicobacter spp. ( Helicobacter The abundance of *Methanobacterium* spp. increased significantly, with *Bacillus brevis* (*Bacillus* spp.) Methanobrevibacter The abundance was significantly reduced. P <0.05), indicating that Salmonella infection can induce the enrichment of pathogenic bacteria and the reduction of beneficial symbiotic bacteria in the cecum of laying hens, thus disrupting the intestinal microecological balance.

[0067] Depend on Figure 4 It can be seen that, compared with the NC group, the GLP group of Bacteroides spp. ( Bacteroides Prevotaceae Ga6A1 group ( Prevotellaceae_Ga6A1_grouP ), Parabacteroides ( Parabacteroides The abundance of beneficial bacteria with polysaccharide metabolites, such as CHKCI001 and *Faecalibacterium*, was significantly increased. Faecalibacterium The abundance of bacteria such as ) was significantly reduced. P <0.05), indicating that the composition of the present invention can selectively enrich the core gut microbiota of carbohydrate metabolism and optimize the basic microbiota structure under healthy conditions.

[0068] Depend on Figure 5 It can be seen that, compared with the PC group, the SGLP group has a higher proportion of candidate Vestibularia species ( Candidatus_Vestibaculum The abundance of *Macrococcus*, *Butylococcus*, and *Sellimonas* significantly increased, while the abundance of these genera significantly decreased. P <0.05), confirming that the composition can reverse the abnormal flora induced by Salmonella challenge and repair the flora disorder in pathological states by regulating the abundance of intestinal symbiotic bacteria.

[0069] 2.7.5 Results of LEfSe Difference Analysis LEfSe analysis was used to screen for differential marker species in the cecal microbiota of each group (LDA threshold > 2), and the results are as follows: Figure 6As shown. Compared with NC, the cecal microbiota structure of the GLP group was significantly altered, with differentially enriched dominant bacteria mainly being Bacteroidetes-associated bacteria, including p__Bacteroidota, c__Bacteroidia, o__Bacteroidales, f__Bacteroidaceae, etc. g__ Bacteroides , s__Bacteroides_barnesiae ,at the same time g__Prevotellaceae_Ga6A1_group The abundance also increased significantly; the NC group was as follows: s__Bacteroides_salanitronis The core differential marker species; the characteristic differentially expressed microbiota of the SGLP group are: g__CHKCI001 .

[0070] 2.7.6 Results of microbial community function prediction (Tax4Fun) Tax4Fun was used to predict KEGG function in the cecal microbiota. The results are shown in [Figure number missing]. Figure 7 The gut microbiota function of all groups of laying hens was mainly based on metabolic and genetic information processing pathways such as carbohydrate metabolism, amino acid metabolism, energy metabolism, and membrane transport. Compared with the NC group, the abundance of carbohydrate and energy metabolism pathways in the GLP group was increased. Salmonella challenge altered the functional structure of the gut microbiota, but the dietary supplementation composition could reverse the challenge-induced functional disorders, bringing the carbohydrate and energy metabolism pathways in the SGLP group back to normal.

[0071] The above results indicate that the composition of the present invention can improve the intestinal health of laying hens by regulating the metabolic function of intestinal flora, enhancing the ability of carbohydrate decomposition and energy utilization, providing a functional basis for the synthesis of short-chain fatty acids.

[0072] 2.8 Non-targeted metabolomics analysis of cecal contents 2.8.1 Principal Component Analysis (PCA) To investigate the overall effect of the composition of the present invention on the metabolic profile of cecal contents in laying hens, unsupervised principal component analysis was used for sample clustering. The results are as follows: Figure 8 As shown, the control group (NC) and the 600 mg / kg composition group (GLP) exhibited a clear separation trend, with good aggregation within each group. The quality control (QC) samples were also concentrated, indicating that the metabolomics data obtained in this study were stable and reliable. These results demonstrate that dietary supplementation with the composition of this invention can significantly alter the overall metabolic profile of the cecal contents in healthy laying hens, exerting a holistic regulatory effect on the intestinal microbial metabolic environment. 2.8.2 Partial Least Squares Discriminant Analysis (PLS-DA) The supervised PLS-DA model was used to further amplify the metabolic differences between groups. Figure 9It was found that the NC group and the GLP group samples were completely separated with no overlapping areas, and the inter-group discrimination was extremely high. This result validated the analytical conclusions of PCA and further confirmed that the combined intervention was the core factor leading to the differences in metabolites in the cecal contents of laying hens, and that the metabolic characteristics of the cecal microbiota underwent specific changes.

[0073] 2.8.3 Volcano plot analysis of differential metabolites by P The screening criteria were <0.05, FC>1.2, or FC<0.83. Differential metabolites between groups were visualized using a volcano plot. Figure 10 It was found that, compared with the NC group, the GLP group screened out 160 significantly different metabolites, of which 79 metabolites were significantly upregulated, 81 metabolites were significantly downregulated, and the remaining metabolites showed no significant changes. These results indicate that the composition of the present invention can broadly regulate the expression levels of microbial metabolites in the cecal contents of laying hens, and bidirectionally regulate intestinal microecology and metabolic homeostasis.

[0074] 2.8.4 Screening and Identification of Significantly Differential Metabolites Structural identification was performed on the screened differential metabolites. After removing unnamed metabolites, 11 significantly differentially expressed metabolites were identified. Table 11 shows that, compared with the control group, the addition of 600 mg / kg of the composition to the diet significantly reduced the levels of seven metabolites in the cecal contents of laying hens, including (2S)-2-butanol glycoside, allantoin, hydroxypioglitazone, and (+)-menthol. P <0.05); the levels of four metabolites, including prostaglandin F1α phosphatidic acid, N-acetyl-7-O-acetylneuraminic acid, and stilbene glycoside, were significantly increased. P <0.05). The aforementioned differential metabolites mainly include lipids, amino acids, flavonoids, and glycosides, which are the core markers for regulating the metabolism of laying hens in the composition of this invention.

[0075] Table 11. Significant differences in metabolite screening results between the 600 mg / kg composition group and the control group in the cecal contents of laying hens. 2.8.5 KEGG pathway functional enrichment analysis To clarify the biological functions of differentially metabolites, KEGG signaling pathway enrichment analysis was performed. Figure 11It was found that the differentially metabolites were significantly enriched in core gut microbial metabolic pathways such as sphingolipid metabolism, nucleotide metabolism, tryptophan metabolism, bile secretion, and amino acid metabolism, as well as in nutrient absorption and immune regulation pathways such as protein digestion and absorption, α-linolenic acid metabolism, and ABC transporters. These results indicate that the composition of the present invention mainly improves the efficiency of nutrient utilization in the laying hen's gut, regulates intestinal immune and anti-inflammatory functions, and maintains gut microbiota homeostasis under healthy conditions by regulating cecal microbiota-mediated amino acid, lipid, and nucleotide metabolic pathways.

[0076] 2.9 Short-chain fatty acids in cecal contents The effects of dietary supplementation with the composition of this invention on the concentration of short-chain fatty acids in the cecum of laying hens are shown in Table 12. Independent samples t-test results showed that, compared with the blank control group, the concentrations of butyric acid and valerate in the cecum of the composition group were significantly increased (…). P <0.05), the concentrations of propionic acid and isohexanoic acid showed an increasing trend (0.05 < P <0.10), there were no significant differences in the concentrations of acetic acid, isobutyric acid, isovaleric acid, and hexanoic acid. P >0.05).

[0077] The above results are highly consistent with the results of jejunal gene expression, 16S rRNA sequencing and functional prediction. This composition enhances the carbohydrate metabolism function of the gut microbiota by enriching beneficial bacteria such as Bacteroides, and promotes the synthesis of short-chain fatty acids (butyric acid and valerate) in the cecal core. This provides a metabolic basis for activating the jejunal GPR41 / GPR43 receptor signaling pathway, and confirms the regulatory effect of the composition of the present invention on the intestinal microecology and metabolism of laying hens.

[0078] Table 12 Effects of dietary supplementation composition on short-chain fatty acid levels in the cecum of Salmonella-infected laying hens The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The use of a composition in the preparation of a product for alleviating Salmonella infection, characterized in that, The composition comprises the following components in parts by weight: 50-70 parts of Ganoderma lucidum polysaccharide, 25-35 parts of Jerusalem artichoke polysaccharide, 25-35 parts of shiitake mushroom polysaccharide, 15-25 parts of Polygonatum odoratum powder, and 15-35 parts of Platycladus orientalis leaf.

2. The application as described in claim 1, characterized in that, The composition comprises the following components in parts by weight: 60 parts of Ganoderma lucidum polysaccharide, 30 parts of Jerusalem artichoke polysaccharide, 30 parts of shiitake mushroom polysaccharide, 20 parts of Polygonatum odoratum powder, and 20 parts of Platycladus orientalis leaf.

3. The application according to claim 1, characterized in that, The product is a feed additive.

4. The application according to claim 3, characterized in that, The feed additive is a feed additive for laying hens.

5. The application according to claim 1, characterized in that, The product is a veterinary drug.

6. A product for alleviating Salmonella infection, characterized in that, The active ingredients include a composition comprising the following components in parts by weight: 50-70 parts of Ganoderma lucidum polysaccharide, 25-35 parts of Jerusalem artichoke polysaccharide, 25-35 parts of lentinan, 15-25 parts of Polygonatum odoratum powder, and 15-35 parts of Platycladus orientalis leaf.

7. The product according to claim 6, characterized in that, The composition comprises the following components in parts by weight: 60 parts of Ganoderma lucidum polysaccharide, 30 parts of Jerusalem artichoke polysaccharide, 30 parts of shiitake mushroom polysaccharide, 20 parts of Polygonatum odoratum powder, and 20 parts of Platycladus orientalis leaf.

8. The product according to claim 7, characterized in that, The product is a feed additive.

9. The product according to claim 8, characterized in that, The feed additive is a feed additive for laying hens.

10. The product according to claim 7, characterized in that, The product is a veterinary drug.

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