Zinc oxide reduction substitution composite additive for improving intestinal health of piglets and application of zinc oxide reduction substitution composite additive

Abstract

The invention discloses a zinc oxide decrement substitution composite additive for improving intestinal health of piglets and application of the zinc oxide decrement substitution composite additive, and belongs to the technical field of animal feed. The zinc oxide decrement substitution composite additive is composed of curcumin, eutectic essential oil, leucine, a microbial agent and an enzyme preparation. The invention provides a zinc oxide decrement substitution composite additive which has the effects of improving intestinal health of piglets and reducing diarrhea rate of the piglets. The compound additive is composed of plant extracts, amino acid, a microbial agent, an enzyme preparation and the like, and all the components are combined for use, so that the weight of piglets can be remarkably increased, the feed utilization rate is increased, and the diarrhea rate of the piglets is reduced. The composite additive disclosed by the invention can realize zinc oxide reduction and replacement, reduces metal discharge at a breeding end, and accords with an environmental protection policy; based on natural components, no residual risk exists, food safety is guaranteed, and sustainable development of the breeding industry is assisted.

Description

Technical Field

[0001] This invention relates to the field of animal feed technology, and in particular to a zinc oxide reduction-substituent compound additive for improving the intestinal health of piglets and its application. Background Technology

[0002] In modern pig farming, the healthy growth of piglets is crucial, and intestinal health is a key factor affecting piglet growth performance and survival rate. During the piglet stage, their gastrointestinal tract is not yet fully developed, their intestinal barrier function is weak, and their intestinal flora is not yet fully stable. This makes piglets extremely sensitive to various stress factors, easily leading to intestinal diseases, affecting liver function, and thus hindering growth and development.

[0003] For a long time, zinc oxide has been widely used in piglet feed due to its properties such as astringing the intestines, regulating immunity, and inhibiting pathogenic microorganisms. It has played a crucial role in controlling diarrhea, promoting growth, and ensuring the intestinal health of piglets. However, with the development of the livestock industry and increased attention to environmental protection and food safety, the drawbacks of high-dose zinc oxide have become increasingly apparent. From an environmental perspective, traditional zinc oxide has low bioavailability, often requiring high doses (pharmacological doses) in practical applications. This results in a large amount of unused zinc being excreted in feces, causing metal accumulation in the soil and polluting the soil ecosystem and surrounding water bodies, which is detrimental to the sustainable development of the livestock industry. From an animal health perspective, high-dose zinc oxide can affect the digestion and absorption of other nutrients in piglets, leading to problems such as pale coat and inhibited protein digestion. It may also alter the intestinal flora structure, increasing the drug resistance of pathogens such as E. coli. Furthermore, zinc accumulates in organs such as the liver, lungs, kidneys, and skin of piglets, posing a potential threat to their health.

[0004] To address these issues, the industry has undertaken numerous explorations. Some studies have attempted to use modified zinc oxide, altering its physicochemical properties to improve bioavailability and reduce dosage. However, the modification process is complex, and the safety of some modified products poses risks; for example, certain silicate mineral-modified zinc oxides exhibit high heavy metal content and unstable quality. Other research focuses on developing novel feed additives, such as probiotics, prebiotics, antimicrobial peptides, organic acids, enzymes, and plant extracts. While these additives can regulate intestinal microecology, enhance digestive function, or improve immunity to some extent, they suffer from limitations such as single-target action and instability, making it difficult to completely replace the comprehensive efficacy of zinc oxide in ensuring the intestinal health of piglets. Therefore, developing an additive and feed that can effectively improve the intestinal health of piglets while achieving reduced zinc oxide dosage has become a crucial issue urgently needing to be addressed in the current pig farming industry. Summary of the Invention

[0005] The purpose of this invention is to provide a zinc oxide reduction and substitution compound additive and its application to improve the intestinal health of piglets, thereby solving the problems existing in the prior art. This invention provides a zinc oxide reduction and substitution compound additive that improves the intestinal health of piglets and reduces the rate of diarrhea. This compound additive is composed of plant extracts, amino acids, microbial agents, and enzyme preparations. The combined use of these components can significantly increase piglet weight, improve feed utilization, and reduce the rate of diarrhea. This compound additive enables the reduction and substitution of zinc oxide, reducing metal emissions from livestock farming and aligning with environmental policies; based on natural ingredients, it poses no residue risk, ensures food safety, and contributes to the sustainable development of the livestock industry.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a zinc oxide reduction-substituent compound additive for improving intestinal health in piglets, which is composed of curcumin, co-crystal essential oil, leucine, bacterial agent and enzyme preparation;

[0008] The eutectic essential oil includes carvacrol, thymol, and L-proline;

[0009] The bacterial agent includes Bacillus subtilis, Pediococcus pentosaceus, and Clostridium butyricum.

[0010] The enzyme preparation includes xylanase, β-mannanase, cellulase, β-glucanase, glucosylamylase, lipase, and protease.

[0011] Furthermore, in the zinc oxide reduction and replacement compound additive, the content of curcumin is 100-200 mg / kg, the content of eutectic essential oil is 150-250 mg / kg, the content of leucine is 500-600 mg / kg, the content of bacterial agent is 150-300 mg / kg, and the content of enzyme preparation is 600-800 mg / kg.

[0012] Furthermore, in the eutectic essential oil, the mass ratio of carvacrol, thymol, and L-proline is 0.5-2:0.5-2:5-7.

[0013] Furthermore, the concentration of Bacillus subtilis cells in the bacterial agent is greater than or equal to 1.9 × 10⁻⁶. 10 CFU / g and Clostridium butyricum cell concentration greater than 5.0 × 10⁻⁶ 8 CFU / g and Pediococcus pentosaceus cell concentration greater than or equal to 1.0 × 10⁻⁶. 9 CFU / g.

[0014] Furthermore, in the enzyme preparation, the xylanase activity is 2 × 10⁻⁶. 7 -4×10 7U / kg, β-mannanase activity is 1×10 7 -3×10 7 U / kg, cellulase activity is 2×10 6 -4×10 6 U / kg, β-glucanase activity is 1×10 6 -3×10 6 The enzyme activity of glucose amylase was 0.5 × 10 U / kg. 7 -2×10 7 U / kg, lipase activity is 1×10 6 -3×10 6 The enzyme activity of the protease was 4 × 10 U / kg. 7 -6×10 7 U / kg.

[0015] The present invention also provides the application of the above-mentioned zinc oxide reduction and replacement compound additive in the preparation of piglet feed that improves the intestinal health of piglets and reduces the rate of diarrhea in piglets.

[0016] The present invention also provides a piglet feed that improves the intestinal health of piglets and reduces the rate of diarrhea in piglets, wherein the piglet feed contains the above-mentioned zinc oxide reduced-volume replacement compound additive.

[0017] Optionally, the zinc oxide reduction and replacement compound additive accounts for 2‰-4‰ of the mass of the piglet feed.

[0018] The present invention discloses the following technical effects:

[0019] This invention provides a zinc oxide reduction and replacement compound additive that improves intestinal health and reduces diarrhea rates in piglets. The compound additive consists of plant extracts, amino acids, microbial agents, and enzyme preparations. The combined use of these components significantly increases piglet weight, improves feed utilization, and reduces diarrhea rates. Experimental verification shows that this compound additive promotes tight junctions of the intestinal epithelium, maintains intestinal mucosal morphology and barrier integrity, strengthens the intestinal physical barrier, reduces the invasion of harmful substances, lowers the risk of intestinal inflammation, and creates a stable environment for intestinal metabolism. This compound additive enables reduced zinc oxide replacement, lowers metal emissions from livestock farming, aligns with environmental policies, and, based on natural ingredients, poses no residue risk, ensuring food safety and contributing to the sustainable development of the livestock industry. Attached Figure Description

[0020] 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.

[0021] Figure 1 The effects of different compound additives on the morphology of piglet jejunal tissue;

[0022] Figure 2 The effects of different compound additives on jejunal villus height, crypt depth, and villus-crypt ratio in piglets;

[0023] Figure 3 Immunofluorescence staining images of Occludin protein in jejunal tissues of piglets from different compound additive groups;

[0024] Figure 4 Immunofluorescence staining images of ZO-1 protein in the jejunal tissue of piglets from different compound additive groups;

[0025] Figure 5 The effects of different compound additives on the expression of Occludin and ZO-1 proteins in the jejunum of piglets;

[0026] Figure 6 The effect of different compound additives on the Alpha diversity index of cecal digesta in piglets;

[0027] Figure 7 This is a PCoA analysis diagram based on the Bray-Curtis distance. Detailed Implementation

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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 apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0032] 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.

[0033] The sources of some of the raw materials involved in the following embodiments of the present invention are as follows:

[0034] Curcumin: Purchased from Guangzhou Kehu Biotechnology Co., Ltd.

[0035] Cocrystal essential oil: a mixture of carvacrol and thymol (1:1) cocrystals with L-proline, with an essential oil content of 25%, purchased from Cocrystal Health Industry (Shanghai) Co., Ltd.

[0036] L-Leucine: Purchased from Zhangjiagang Sipu Biochemical Co., Ltd.

[0037] Microecological preparation: composed of Bacillus subtilis, Pediococcus pentosaceus, Clostridium butyricum, and maifanite, wherein Bacillus subtilis (CFU / g) ≥ 1.9 × 10 10 Clostridium butyricum (CFU / g) > 5.0 × 10⁻⁶ 8 Pediococcus pentosaceus (CFU / g) ≥1.0×10 9 Purchased from Shandong Baolai Lailai Biotechnology Co., Ltd.

[0038] Enzyme preparation: Xylanase (enzyme activity 2.5 × 10⁻⁶) 7 U / kg), β-mannanase (enzyme activity 2×10 7 U / kg), cellulase (enzyme activity 3×10 6 U / kg), β-glucanase (enzyme activity 2×10⁻⁶) 6 U / kg), glucose amylase (enzyme activity 1×10 7 U / kg), lipase (enzyme activity 2×10 6U / kg) and protease (enzyme activity 5×10 7 U / kg), purchased from Shandong Longket Enzyme Preparation Co., Ltd.

[0039] Unless otherwise specified, other experimental materials involved in the following embodiments of the present invention can be purchased through conventional channels; other experimental methods involved, unless otherwise specified, are carried out in accordance with experimental methods known in the art.

[0040] Example 1

[0041] This embodiment provides a zinc oxide reduction alternative compound additive (PBE) composed of curcumin, co-crystal essential oil, leucine, microbial agent, and enzyme preparation. Specifically, curcumin is 150 mg / kg, co-crystal essential oil is 200 mg / kg, and leucine is 550 mg / kg; the microbial agent (200 mg / kg) consists of Bacillus subtilis, Pediococcus pentosaceus, Clostridium butyricum, and maifanite, wherein Bacillus subtilis ≥ 1.9 × 10⁻⁶. 10 CFU / g, Clostridium butyricum >5.0×10 8 CFU / g, Pediococcus pentosaceus ≥1.0×10 9 CFU / g; The enzyme preparation (650 mg / kg) consists of xylanase, β-mannanase, cellulase, β-glucanase, glucosylamylase, lipase, and protease, among which the enzyme activity of xylanase is 2.5 × 10⁻⁶. 7 The enzyme activity of β-mannanase is 2 × 10 U / kg. 7 U / kg, cellulase activity is 3×10 6 U / kg, β-glucanase activity is 2×10 6 U / kg, glucosyl amylase activity is 1×10 7 U / kg, lipase activity is 2×10 6 U / kg and protease activity 5×10 7 U / kg. Mix the above components evenly to obtain the composite additive for reducing zinc oxide dosage.

[0042] Comparative Example 1

[0043] The only difference between the composite additive in this comparative example and Example 1 is that the microbial agent is omitted.

[0044] Comparative Example 2

[0045] The only difference between the composite additive in this comparative example and Example 1 is that the enzyme preparation is omitted.

[0046] Comparative Example 3

[0047] The only difference between the composite additive in this comparative example and Example 1 is that curcumin, co-crystal essential oil and leucine are omitted.

[0048] Comparative Example 4

[0049] The only difference between the composite additive in this comparative example and Example 1 is that Pediococcus pentosaceus and Clostridium butyricum in the bacterial agent are omitted.

[0050] Comparative Example 5

[0051] This comparative example uses a feed additive previously researched by the inventors, as described in Example 1 of patent document CN 118716486 A (publication date: 2024.10.01).

[0052] Comparative Example 6

[0053] This comparative example provides a zinc oxide-tannic acid additive (ZTA) composed of 2000 mg / kg zinc oxide and 1000 mg / kg tannic acid.

[0054] Experimental Example 1

[0055] Piglets were pre-fed at 14 days of age for a 14-day pre-trial period, using piglets with an average weight of (8.02±0.05) kg at 28 days of age. The formal feeding period was 14 days. Piglets were randomly divided into 8 treatments, with 6 replicates per treatment and 6 piglets per replicate. There was no significant difference in initial weight among the treatments (P>0.05). Treatment group 1 served as the control group, fed a basal diet containing montmorillonite (3‰). Treatments 2-8 replaced the montmorillonite in the basal diet with the compound additive (PBE) of Example 1, the additive (PE) of Comparative Example 1, the additive (PB) of Comparative Example 2, the additive (BE) of Comparative Example 3, the additive (PE-1) of Comparative Example 4, the additive (XY) of Comparative Example 5, and the additive (ZTA) of Comparative Example 6, respectively. The feed formulation and the content of its main nutrients are shown in Table 1.

[0056] Table 1. Basal Diet Formulation and Nutrient Levels (Air-dried Basis, %)

[0057]

[0058]

[0059] Note: The premix provides the following per kilogram of feed: Vitamin A 1750 IU; Vitamin D3 150 IU; Vitamin E 11.8 IU; Vitamin K3 0.51 mg; Vitamin B1 0.97 mg; Vitamin B2 2.4 mg; Pantothenic acid 7.62 mg; Niacin 29.89 mg; Pyridoxine 1.39 mg; Biotin 0.05 mg; Folic acid 0.3 mg; Vitamin B... 120.01 mg; Manganese (manganese sulfate monohydrate) 25 mg; Iron (ferrous sulfate monohydrate) 80 mg; Zinc (zinc sulfate monohydrate) 75 mg; Copper (copper sulfate pentahydrate) 18 mg; Iodine (potassium iodate) 0.5 mg; Selenium (sodium selenite) 0.3 mg.

[0060] This experiment was conducted at Anfei Livestock Farm Luozhuang No. 6 in Linyi City. Before the experiment, the pigsties were thoroughly disinfected using 2% sodium hydroxide solution and potassium persulfate solution, respectively. During the experiment, the ambient temperature in the pigsties was maintained at 22-26℃ and the relative humidity at 60%-70%. Piglets had free access to feed and water during the experiment. Feed intake in each replicate pen was recorded daily, and the piglets' mental state and fecal morphology were observed. The experiment consisted of a 14-day pre-trial period and a 14-day formal trial period. Immunization and other procedures were carried out normally. At the end of the formal trial period (14 days), before morning feeding, one piglet close to the average weight was randomly selected from each replicate for slaughter and sampling.

[0061] At the beginning and end of the formal trial period, all weaned piglets were weighed to calculate the average daily gain (ADG). During the trial, feed intake of weaned piglets was recorded for each replicate, and the average daily feed intake (ADFI) and feed-gain ratio (F / G) were calculated. During the trial, the feces of piglets in each pen were observed three times a day (morning, noon, and evening) to determine if they were formed. Piglets with unformed feces and loose stools attached to the anus were considered to have diarrhea. The number of piglets with diarrhea was counted, and the diarrhea rate was calculated.

[0062] Average daily weight gain (ADG) = (final weight at the start of the trial - initial weight) / number of days in the trial.

[0063] Average daily feed intake (ADFI) = (initial feed weight - final feed weight) / number of piglets.

[0064] Feed conversion ratio (F / G) = Average daily feed intake (kg) / Daily weight gain (kg).

[0065] Diarrhea rate = (number of piglets with diarrhea / (number of piglets in the experiment × number of days in the experiment)) × 100%. Number of piglets with diarrhea: the total number of days of diarrhea for all piglets during the experiment (e.g., 1 piglet with diarrhea for 3 days is counted as 3 counts).

[0066] Following the above formula, the average daily weight gain, average daily feed intake, and feed conversion ratio (FCR) per pig were calculated to obtain the results for each group. The effects of different compound additives on piglet growth performance and diarrhea rate are shown in Table 2. Analysis of variance showed that, compared with the control group, all other groups significantly increased the final weight and average daily feed intake of piglets, while significantly decreasing the FCR (P<0.05), and exhibited significant anti-diarrheal effects. Specifically, compared with the zinc oxide-tannic acid group, the compound additive group significantly increased the final weight of piglets (P<0.05) and decreased the FCR (P<0.05). Under the conditions of this experiment, the compound additive group achieved growth-promoting and diarrhea-preventing effects similar to those of zinc oxide-tannic acid.

[0067] Table 2 Effects of different compound additives on growth performance of piglets

[0068]

[0069] Experimental Example 2

[0070] Based on the results of Experiment 1, this experiment examined the apparent nutrient digestibility of the zinc oxide-tannic acid group (ZTA) and the compound additive group (PBE), and their effects on serum metabolic indicators and intestinal health in piglets. The specific procedures and results are as follows:

[0071] 1. Apparent digestibility of nutrients

[0072] Three days before the end of the experiment, clean feces were collected for three consecutive days. The fecal samples were mixed evenly, and 1 / 5 of the total fecal sample was taken and acid was added (10 mL of 10% sulfuric acid per 100 g of fresh feces) to fix nitrogen. This was used to determine the crude protein content in the feces based on the fresh sample. The remaining 2 / 5 of the fresh fecal sample was dried at 65℃ to prepare air-dried fecal samples. The apparent nutrient digestibility was determined using the endogenous indicator method (Acid-insoluble ash, AIA) in this experiment.

[0073] AIA content determination: Approximately 1g of sample was accurately weighed into a 5cm × 6cm polyester mesh bag of known mass and sealed using a plastic sealer. The sample was added to a beaker containing 3mol / L hydrochloric acid, heated to boiling, and maintained at a gentle boil for 30 minutes. The sample was then removed, washed, and the water inside the bag was squeezed out. The bag was then placed in a 105℃ oven and dried for 30 minutes. The dried sample was placed in a crucible and first carbonized on an electric furnace until smokeless, then calcined in a muffle furnace at 550℃ for 1 hour. The AIA content in each sample bag was then determined.

[0074] Determination of Nutrient Content in Feed and Manure: The determination of dry matter (DM), crude protein (CP), and crude fat (EE) in feed and manure samples was performed according to AOAC (2012) and national standards. Dry matter was determined using the drying method at (103±2℃) (GB / T 6435-2014), crude protein was determined using the semi-micro Kjeldahl method (GB / T 6432-2018), crude fat was determined using the Soxhlet ether extraction method (GB / T 6433-2006), and crude ash was determined using the ignition method (GB / T 6438-2007). Organic matter (OM) was calculated using the formula: OM (%) = DM (%) - CA (%). Apparent nutrient digestibility was calculated using the following formula:

[0075]

[0076] The effects of different compound additives on the apparent digestibility of nutrients in piglets are shown in Table 3. Analysis of variance showed that, compared with the control group (CON), the apparent digestibility of dry matter, organic matter, crude protein, and crude fat in piglets in the zinc oxide-tannic acid group and the plant extract-bacteria-enzyme group was significantly increased (P<0.05).

[0077] Table 3. Effects of different compound additives on apparent nutrient digestibility in piglets (%)

[0078]

[0079] 2. Serum enzymes, metabolites, and antioxidants

[0080] At the end of the formal experiment (14 days) before early feeding, 5 mL of blood was collected from one piglet in each replicate using a vacuum coagulation tube via the anterior vena cava method. The blood collection tubes were left at room temperature for 15 min, then centrifuged (3,500 rpm, 15 min). Serum was collected, aliquoted, and stored at -80°C for subsequent analysis. Serum biochemical parameters were measured using a COBUS MIRAPlus fully automated biochemical analyzer (Roche, USA). Serum enzymes include alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH); serum metabolites include total protein (TP), albumin (ALB), blood urea nitrogen (SUN), triglycerides (TG), total cholesterol (TCHO), high-density lipoprotein (HDL), low-density lipoprotein (LDL), and serum calcium (CA).

[0081] 2.1 Serum enzymes

[0082] The effects of different compound additives on serum enzyme levels in piglets are shown in Table 4. Analysis of variance showed that, compared with the control group, the activities of aspartate aminotransferase (AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) in serum were significantly increased in the zinc oxide-tannic acid group (P<0.05), while only ALP activity was significantly increased in the compound additive group (P<0.05). Compared with the zinc oxide-tannic acid group, the activities of AST, ALP, ALP, and LDH in serum were significantly decreased in the compound additive group (P<0.05). Other activities showed no significant differences (P>0.05).

[0083] Table 4. Effects of different compound additives on serum enzymes in piglets (U / L)

[0084]

[0085] 2.2 Serum metabolites

[0086] The effects of different compound additives on the content of serum metabolites in piglets are shown in Table 5. Compared with the control group, the zinc oxide-tannic acid group showed significantly higher levels of serum triglycerides, total cholesterol, and high-density lipoprotein (P<0.05), while the compound additive group showed significantly higher levels of serum total protein, albumin, high-density lipoprotein, and serum calcium (P<0.05). Compared with the zinc oxide-tannic acid group, the compound additive group showed significantly higher levels of serum total protein and albumin (P<0.05), while the compound additive group showed significantly lower levels of serum triglycerides and high-density lipoprotein (P<0.05). There were no significant effects among the treatment groups on other indicators (P>0.05).

[0087] Table 5. Effects of different compound additives on serum metabolites in piglets

[0088]

[0089] 2.3 Serum antioxidant capacity

[0090] The antioxidant indicators of different compound additives on piglet serum are shown in Table 6. Compared with the control group, the SOD activity of the zinc oxide-tannic acid group was significantly increased (P<0.05), while the MDA content of both the zinc oxide-tannic acid group and the compound additive group was significantly decreased (P<0.05). There were no significant differences in serum GSH-PX activity and T-AOC among the groups (P>0.05).

[0091] Table 6. Effects of different compound additives on serum antioxidant indicators in piglets (U / L)

[0092]

[0093]

[0094] Note: SOD: Superoxide dismutase; GSH-PX: Glutathione peroxidase; T-AOC: Total antioxidant capacity; MDA: Malondialdehyde.

[0095] 3. Jejunal health

[0096] 3.1 Antioxidant capacity of the jejunum

[0097] Table 7 shows the antioxidant indices of different compound additives on the jejunum of piglets. It can be seen that, except for the compound additive group, where the GSH-PX activity in the jejunum was significantly higher than that in the control group and the zinc oxide-tannic acid group (P<0.05), the other indices of the compound additive group were not significantly different from those of the zinc oxide-tannic acid group (P>0.05).

[0098] Table 7. Effects of different compound additives on antioxidant indices of piglet jejunum (U / L)

[0099]

[0100] Note: SOD: Superoxide dismutase; GSH-PX: Glutathione peroxidase; T-AOC: Total antioxidant capacity; MDA: Malondialdehyde.

[0101] 3.2 Histological morphology of the jejunum

[0102] The effects of different compound additives on the morphological development of piglet jejunal tissue are shown in the figure. Figure 1 and Figure 2 In the control group and the zinc oxide-tannic acid group, the jejunal villi of piglets were sparsely arranged, with incomplete epithelium and some shedding; lymphocytes were increased in the intestinal glands. In the compound additive group, the jejunal villi were densely arranged, with no shedding or lymphocyte infiltration. Compared with the control group and the zinc oxide-tannic acid group, the villi height in the compound additive group was significantly increased (P<0.05). Compared with the control group, the crypt depth in the zinc oxide-tannic acid group was significantly decreased (P<0.05), while the villi-crypt ratio in both the zinc oxide-tannic acid group and the compound additive group was significantly increased (P<0.05).

[0103] 3.3 Jejunal barrier

[0104] The effects of different compound additives on the expression of tight junction proteins in the jejunum of piglets are shown in the figure. Figures 3-5 Compared with the control group, the expression of Occludin (red) and ZO-1 protein (green) in the jejunum of the zinc oxide-tannic acid group and the compound additive group was significantly increased (P<0.05). The expression of Occludin and ZO-1 protein in the jejunum of the compound additive group was significantly higher than that of the zinc oxide-tannic acid group (P<0.05). There were no significant differences among the other groups (P>0.05).

[0105] 4. Cecal microbial metagenomic analysis

[0106] 4.1 Statistical analysis of metagenomic sequencing data of cecal chyme

[0107] Metagenomic sequencing was performed on cecal digesta samples from 18 piglets using the Illumina NovaSeq 6000 sequencing platform. The raw data statistics are shown in Table 8. A total of 128.09 G of filtered data (Cleanbases) was generated from the 18 cecal digesta samples. The average data volume per sample was 7.485 G, 7.105 G, and 6.945 G for the control group (CON), zinc oxide-tannic acid group (ZTA), and compound additive group (PBE), respectively. The proportions of Q20 and Q30 were above 99% and 96%, respectively, and the base percentages ranged from 41.57% to 47.93%, with an effective data rate exceeding 98%. The results indicate that the filtered data quality is good and can be used for subsequent analysis.

[0108] Table 8. Statistics of Metagenomic Sequencing Data

[0109]

[0110] 4.2 Cecal chyme metagenomic assembly results

[0111] After preprocessing, clean data was obtained and assembled using MEGAHIT assembly software. For the scaftigs generated from each sample, fragments smaller than 500 bp were filtered out, and statistical analysis and subsequent gene prediction were performed. The metagenomic assembly results are shown in Table 9. The average length of N50 was 1296.72 bp; the average length of N90 was 574.33 bp.

[0112] Table 9. Statistics on metagenomic assembly results

[0113]

[0114] 4.3 Results of metagenomic gene prediction for cecal chyme

[0115] After using MetaGeneMark to predict the ORFs of scaftigs (≥500bp) for each sample and removing redundancy, the basic information of the gene predictions is shown in Table 10. A total of over 4 million unique gene counts (ORFs) were obtained. Among these, genes containing both start and stop codons accounted for 34.5%, those without both start and stop codons accounted for 14.02%, those with only start codons accounted for 27.7%, and those with only stop codons accounted for 23.77%. The total gene length was 2567.43 Mbp; the average length was 581.96 bp; and the GC content was 45.42%.

[0116] Table 10: Basic Information Statistics of Gene Catalog

[0117]

[0118]

[0119] 4.4 Analysis of microbial species diversity in cecal digesta

[0120] Depend on Figure 6 As shown, alpha diversity analysis based on species abundance at the genus level revealed that, compared with the control group, the zinc oxide-tannic acid group and the compound additive group showed significantly increased microbial diversity (Simpson index) (P<0.05), while microbial richness (ACE and Chao1 index) showed no significant differences among the treatment groups (P>0.05). To investigate the effects of different compound additives on the cecal microbial community structure, principal coordinate analysis (PCoA) was performed at the phylum and genus levels. Figure 7As shown, at the phylum level, the contribution rates of the first principal coordinate (PCoA1) and the second principal coordinate (PCoA2) were 56.8% and 22.25%, respectively (top figure), indicating significant separation among the treatment groups in two-dimensional space. Structural differences existed between the control group (CON) and the two additive groups (zinc oxide-tannic acid group and composite additive group), with the zinc oxide-tannic acid group exhibiting a more distant distribution, suggesting a potentially different regulatory mechanism. At the genus level (bottom figure), the contribution rates of the first principal coordinate (PCoA1) and the second principal coordinate (PCoA2) were 49.43% and 19.28%, respectively. The control group remained significantly separated from the other groups, indicating that the microbial structure at the genus level was also regulated. Overall, the samples from each treatment group showed both overlapping and separated portions in coordinate space. This suggests that while maintaining similarity, certain changes occurred in the microbial community. The composite additive significantly altered the composition of the cecal microbiota, significantly changing the β-diversity of the microbial community, and the community differentiation characteristics at the genus level were more clearly defined.

[0121] 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. A zinc oxide reduction-substituent compound additive for improving intestinal health in piglets, characterized in that, It is composed of curcumin, co-crystal essential oil, leucine, bacterial agent and enzyme preparation; The eutectic essential oil includes carvacrol, thymol, and L-proline; The bacterial agent includes Bacillus subtilis, Pediococcus pentosaceus, and Clostridium butyricum. The enzyme preparation includes xylanase, β-mannanase, cellulase, β-glucanase, glucosylamylase, lipase, and protease.

2. The zinc oxide reduction and replacement composite additive according to claim 1, characterized in that, The zinc oxide reduction and replacement compound additive contains curcumin at a content of 100-200 mg / kg, eutectic essential oil at a content of 150-250 mg / kg, leucine at a content of 500-600 mg / kg, bacterial agent at a content of 150-300 mg / kg, and enzyme preparation at a content of 600-800 mg / kg.

3. The zinc oxide reduction and replacement composite additive according to claim 1, characterized in that, In the eutectic essential oil, the mass ratio of carvacrol, thymol, and L-proline is 0.5-2:0.5-2:5-7.

4. The zinc oxide reduction and replacement composite additive according to claim 1, characterized in that, The bacterial agent contains Bacillus subtilis cells at a concentration greater than or equal to 1.9 × 10⁻⁶. 10 CFU / g and Clostridium butyricum cell concentration greater than 5.0 × 10⁻⁶ 8 CFU / g and Pediococcus pentosaceus cell concentration greater than or equal to 1.0 × 10⁻⁶. 9 CFU / g.

5. The zinc oxide reduction and replacement composite additive according to claim 1, characterized in that, In the enzyme preparation, the xylanase activity is 2 × 10⁻⁶. 7 -4×10 7 U / kg, β-mannanase activity is 1×10 7 -3×10 7 U / kg, cellulase activity is 2×10 6 -4×10 6 U / kg, β-glucanase activity is 1×10 6 -3×10 6 The enzyme activity of glucose amylase was 0.5 × 10 U / kg. 7 -2×10 7 U / kg, lipase activity is 1×10 6 -3×10 6 The enzyme activity of the protease was 4 × 10 U / kg. 7 -6×10 7 U / kg.

6. The application of the zinc oxide reduction and substitution compound additive according to any one of claims 1-5 in the preparation of piglet feed that improves intestinal health and reduces the rate of diarrhea in piglets.

7. A piglet feed that improves intestinal health and reduces diarrhea rates in piglets, characterized in that, The piglet feed contains the zinc oxide reduction and replacement compound additive as described in any one of claims 1-5.

8. The piglet feed according to claim 7, characterized in that, The zinc oxide reduction and replacement compound additive accounts for 2‰-4‰ of the mass of the piglet feed.

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