Natural plant feed additive for replacing antibiotics in broiler chicken diet and application thereof

Abstract

This application belongs to the field of broiler feed processing technology, specifically relating to a natural plant-based feed additive that can replace antibiotics in broiler diets and its application. This natural plant-based feed additive includes tea saponin extract and Pueraria lobata-Scutellaria baicalensis decoction extract; the mass ratio of tea saponin extract to Pueraria lobata-Scutellaria baicalensis decoction extract is 3-9:1; the Pueraria lobata-Scutellaria baicalensis decoction extract is an extract of Pueraria lobata, Scutellaria baicalensis, Coptis chinensis, and Glycyrrhiza uralensis. This invention uses a compound of Pueraria lobata-Scutellaria baicalensis decoction extract and tea saponin as a broiler feed additive. This green compound is rich in various effective ingredients, which can improve broiler growth performance and intestinal health, avoid the addition of antibiotics, promote the sustainable development of livestock and poultry farming, and is a relatively safe feed additive with no toxic side effects.

Description

Technical Field

[0001] This application belongs to the field of broiler feed processing technology, specifically relating to a natural plant feed additive that can replace antibiotics in broiler diets and its application. Background Technology

[0002] Currently, with the rapid development of the livestock industry, poultry health has become an increasingly important topic. In chicken farming, some pathogens can harm chickens' health, causing significant economic losses when outbreaks occur on a large scale, making prevention particularly crucial. The intestines are one of the largest and most complex organs in chickens, and their health plays a vital role in defending against pathogens and digesting and absorbing feed. Once intestinal health is compromised, the chicken's life and production activities will be affected, and most diseases are directly or indirectly related to intestinal problems. The most effective way to prevent intestinal diseases in chickens is by adding antibiotics; however, the problems of antibiotic resistance and chicken food safety caused by long-term, high-dose antibiotic use are becoming increasingly prominent. Therefore, there is an urgent need to develop green and eco-friendly antibiotic alternatives to achieve the healthy development of the livestock industry. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide a natural plant-based feed additive that can replace antibiotics in broiler diets and its application. Specifically, the following technical solution is adopted:

[0004] In a first aspect, the present invention provides a natural plant feed additive that can replace antibiotics in broiler diets, characterized in that the natural plant feed additive includes tea saponin extract and kudzu root and scutellaria root decoction extract; the mass ratio of tea saponin extract to kudzu root and scutellaria root decoction extract is 3~9:1; and the kudzu root and scutellaria root decoction extract is an extract of kudzu root, scutellaria root, coptis root and prepared licorice root.

[0005] In this invention, tea saponins and kudzu root and scutellaria decoction extracts, as natural plant active ingredients, can promote the growth of beneficial bacteria, inhibit the reproduction of harmful bacteria, maintain the balance of intestinal microecology, improve the body's resistance, prevent pathogen invasion, maintain the relative stability of the intestinal microbial ecosystem, promote the healthy growth of livestock and poultry, and improve growth performance.

[0006] As a further preferred embodiment, the extract of Ge Gen Qin Lian Tang is obtained by the following extraction method:

[0007] Kudzu root, Scutellaria baicalensis, Coptis chinensis and prepared licorice root were pulverized and mixed. Then, ultrasonic water extraction was carried out at a material-to-liquid ratio of 1:5~7 and a temperature of 50 ℃~60 ℃. After filtration, water extract and residue were obtained.

[0008] The dregs were subjected to ultrasonic alcohol extraction at a material-to-liquid ratio of 1:3 to 5, and then filtered to obtain an alcohol extract.

[0009] The aqueous extract and alcoholic extract were concentrated and dried to obtain aqueous extract and alcoholic extract, respectively. Finally, the aqueous extract and alcoholic extract were mixed to obtain the Ge Gen Qin Lian Tang extract.

[0010] As a further preferred embodiment, the mass ratio of kudzu root, scutellaria root, coptis root, and prepared licorice root is 4.5~5.5:1.6~2.0:1.6~2.0:1.0~1.5.

[0011] As a further preferred embodiment, the mass ratio of water extract to alcohol extract in the Ge Gen Qin Lian Tang extract is 40%~50%:60%~50%.

[0012] As a further preferred embodiment, the tea saponin extract is obtained by the following extraction method:

[0013] Tea seed cake was pulverized to a particle size of 150μm~250μm, dried, and subjected to ultrasonic extraction with ethanol or methanol as the extraction solvent at a material-to-liquid ratio of 1:4~8 and a temperature of 30℃. The extract was then concentrated and dried to obtain tea saponin extract.

[0014] As a further preferred embodiment, both ultrasonic water extraction and ultrasonic alcohol extraction employ alternating concentrated and divergent ultrasound. Each concentrated ultrasound session lasts 10-15 seconds, and each divergent ultrasound session lasts 5-10 seconds. The concentrated extraction power is 1200 W-3000 W, and the divergent extraction power is 300 W-1000 W. Ultrasonic technology offers high extraction efficiency for tea saponins. The cavitation effect generated when ultrasound acts on the liquid material instantly disrupts the cell walls of tea seeds. Simultaneously, the vibrations enhance the release, diffusion, and dissolution of tea saponins while maintaining their biological activity, significantly improving the extraction efficiency of tea saponins. Furthermore, the extraction time is short, reducing energy consumption.

[0015] Green and environmentally friendly: No chemical solvents are used, avoiding the environmental pollution problems caused by organic solvent extraction. It is green and environmentally friendly, and the extract can be repeatedly recycled, allowing for the full utilization and development of camellia oil cake resources. Low equipment requirements, easy to industrialize.

[0016] As a further preferred embodiment, the extraction time for both ultrasonic water extraction and ultrasonic alcohol extraction is 40 min to 60 min.

[0017] As a further preferred embodiment, the concentrated product is dried by spray drying, with specific conditions of 120°C air intake and 85°C air outlet.

[0018] Secondly, the present invention provides a method for using the above-mentioned natural plant feed additive that replaces antibiotics in broiler feed, wherein 1.3g to 3.0g of the natural plant feed additive is added to every 1kg of broiler feed.

[0019] Thirdly, the present invention provides the application of the above-mentioned natural plant feed additives in feeds that improve the intestinal health of broilers.

[0020] The beneficial effects of this invention are as follows:

[0021] This invention provides a feed additive composed of tea saponin and kudzu root and scutellaria decoction extract, which can improve the growth performance of broilers and improve intestinal health. It is a green and safe feed additive that is non-toxic, has no side effects, does not pollute the environment, and is in line with sustainable development. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying 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.

[0023] Figure 1 The figures show growth performance indicators;

[0024] Figure 2 The image shows the morphological parameters of the duodenal intestinal tract at 24 days.

[0025] Figure 3 The image shows the morphological parameters of the jejunum at 24 days.

[0026] Figure 4 The image shows the morphological parameters of the ileum at 24 days.

[0027] Figure 5 The image shows the morphological parameters of the duodenal intestinal tract at 43 days.

[0028] Figure 6 The image shows the morphological parameters of the jejunum at 43 days old.

[0029] Figure 7 The image shows the morphological parameters of the ileum at 43 days old.

[0030] Figure 8 The figure shows the expression levels of intestinal barrier-related genes in the ileum at 43 days.

[0031] Figure 9 The figure shows the expression levels of intestinal barrier-related proteins in the ileum at 43 days.

[0032] Figure 10 The image shown is an immunohistochemical result of Occludin protein.

[0033] Figure 11 The image shown is an immunohistochemical result of Claudin-1 protein.

[0034] Figure 12 The image shown is an immunohistochemical result of ZO-1 protein.

[0035] Figure 13 The figures show serum antioxidant indices at 24 and 43 days.

[0036] Figure 14 The figure shows the α-diversity index;

[0037] Figure 15 The figure shows the β-diversity index;

[0038] Figure 16 The figure shows the abundance analysis at the phylum level;

[0039] Figure 17 The figure shows the abundance analysis at the genus level;

[0040] Figure 18 The figure shows the prediction of species abundance and pathogenic species phenotypes. Detailed Implementation

[0041] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0042] Example 1

[0043] A natural plant-based feed additive to replace antibiotics in broiler diets, the preparation method of which includes the following steps:

[0044] (1) Extraction of tea saponins:

[0045] Tea dregs were crushed to a particle size of 200 μm using a traditional Chinese medicine pulverizer and placed in a 60℃ oven to remove moisture. 75% (v / v) ethanol was used as the extraction solvent, and the extraction conditions were: material-to-liquid ratio of 1:5 (g / mL), extraction temperature of 30℃, extraction time of 30 min, concentrated extraction power of 1200 W, and divergent extraction power of 300 W. The extract was concentrated to a dry matter content of about 50%, and then spray-dried with air inlet at 120℃ and air outlet at 85℃. The yield of tea saponins was 38%, and the purity was 57%.

[0046] (2) Preparation of Gegen Qinlian Decoction Extract:

[0047] Kudzu root, Coptis chinensis, Scutellaria baicalensis, and prepared licorice root were pulverized and mixed in a mass ratio of 5.5:2:2:1.5. The mixture was then subjected to ultrasonic water extraction once, with a material-to-liquid ratio of 1:5 (g / mL), at 55℃ for 50 min, followed by filtration. The residue was then subjected to ultrasonic ethanol extraction once more, using 75% ethanol at a material-to-liquid ratio of 1:3.5 (g / mL) for 40 min. Both ultrasonic extractions employed alternating focused and divergent ultrasound, with each focused ultrasound session lasting 12 s and divergent ultrasound lasting 5 s. The focused extraction power was 1500 W, and the divergent extraction power was 500 W. The extracts were then concentrated and dried at 70℃, yielding two extracts with water extraction rates of 17% and ethanol extraction rates of 12%. The water and ethanol extracts were then mixed at a mass ratio of 50% and 50%, respectively, to obtain the traditional Chinese medicine extract (Kudzu Root and Scutellaria Decoction Extract).

[0048] (3) The tea saponin extract and the kudzu root and scutellaria decoction extract were mixed at a mass ratio of 75%:25% to obtain a natural plant feed additive.

[0049] Example 2

[0050] A natural plant-based feed additive to replace antibiotics in broiler diets, the preparation method of which includes the following steps:

[0051] (1) Extraction of tea saponins:

[0052] Tea dregs were crushed to a particle size of 200 μm using a traditional Chinese medicine pulverizer and placed in a 60℃ oven to remove moisture. 75% (v / v) ethanol was used as the extraction solvent, and the extraction conditions were: material-to-liquid ratio of 1:6 (g / mL), extraction temperature of 30℃, extraction time of 30 min, concentrated extraction power of 1500 W, and divergent extraction power of 500 W. The extract was concentrated to a dry matter content of about 50% and then spray-dried with air inlet at 120℃ and air outlet at 85℃. The yield of tea saponins was 40%, and the purity was 54%.

[0053] (2) Preparation of Gegen Qinlian Decoction Extract:

[0054] Kudzu root, Coptis chinensis, Scutellaria baicalensis, and prepared licorice root were pulverized and mixed in a mass ratio of 5:2:2:1. The mixture was then subjected to ultrasonic water extraction once, with a material-to-liquid ratio of 1:6 (g / mL), at 50℃ for 60 min, followed by filtration. The residue was then subjected to ultrasonic ethanol extraction once more, using 70% ethanol at a material-to-liquid ratio of 1:4 (g / mL) for 50 min. Both ultrasonic extractions employed alternating focused and divergent ultrasound, with each session consisting of 15 seconds of focused ultrasound followed by 8 seconds of divergent ultrasound. The focused extraction power was 2000 W, and the divergent extraction power was 800 W. The extracts were then concentrated and dried at 75℃, yielding two extracts with water extraction rates of 18% and ethanol extraction rates of 15%. The water and ethanol extracts were then mixed at 45% and 55% respectively to obtain the herbal extract (Kudzu Root and Scutellaria baicalensis Decoction Extract).

[0055] (3) The tea saponin extract and the kudzu root and scutellaria decoction extract were mixed at a mass ratio of 75%:25% to obtain a natural plant feed additive.

[0056] Example 3

[0057] A natural plant-based feed additive to replace antibiotics in broiler diets, the preparation method of which includes the following steps:

[0058] (1) Extraction of tea saponins:

[0059] Tea dregs were crushed to a particle size of 200 μm using a traditional Chinese medicine pulverizer and placed in a 60℃ oven to remove moisture. 75% (v / v) ethanol was used as the extraction solvent, with an extraction condition of a solid-liquid ratio of 1:8 (g / mL), an extraction temperature of 30℃, an extraction time of 30 min, a concentrated extraction power of 3000 W, and a divergent extraction power of 1000 W. The extract was concentrated to a dry matter content of approximately 50% and then spray-dried at 120℃ inlet and 85℃ outlet. The yield of tea saponins was 44%, and the purity was 50%.

[0060] (2) Preparation of Gegen Qinlian Decoction Extract:

[0061] Kudzu root, Coptis root, Scutellaria root, and prepared licorice root were pulverized and mixed in a mass ratio of 4.6:1.5:2:1.4. The mixture was then subjected to ultrasonic water extraction once, with a material-to-liquid ratio of 1:7 (g / mL), at 60℃ for 40 min, followed by filtration. The residue was then subjected to ultrasonic ethanol extraction once more, using 70% ethanol at a material-to-liquid ratio of 1:3 (g / mL) for 60 min. Both ultrasonic extractions employed alternating focused and divergent ultrasound, with each session consisting of 15 s of focused ultrasound followed by 10 s of divergent ultrasound. The focused extraction power was 1200 W, and the divergent extraction power was 1000 W. The extracts were then concentrated and dried at 80℃, yielding two extracts with water extraction rates of 17% and ethanol extraction rates of 14.5%. The water and ethanol extracts were then mixed at 40% and 60% respectively to obtain the herbal extract (Kudzu Root and Scutellaria Decoction Extract).

[0062] (3) The tea saponin extract and the kudzu root and scutellaria decoction extract were mixed at a mass ratio of 75%:25% to obtain a natural plant feed additive.

[0063] Example 4

[0064] The efficacy of natural plant-based feed additives as alternatives to antibiotics in broiler diets was investigated experimentally, as detailed below:

[0065] 1.1 Experimental Materials and Grouping

[0066] Forty-three hundred and thirty-two healthy one-day-old broiler chickens with no significant difference in weight were selected and pre-fed for three days. They were then randomly divided into six groups according to the example, with six replicates per group and twelve chickens per replicate. The specific groups were Example 1, Example 2, Example 3, Example 4, Example 5, and Example 6. The experiment lasted for 43 days. Tongwei stage feed was used as the experimental diet. One chicken was randomly selected from each replicate for slaughter at 24 days and 43 days.

[0067] The animal experiment was conducted at Jiabo Biotechnology Company in Duchang County from August 18, 2024 to September 30, 2024. The experimental chickens were raised in net cages, and the environment and feeding methods were consistent with those of the experimental chickens. They had free access to feed, received routine immunizations, and cleaning and disinfection were carried out in accordance with normal feeding methods.

[0068] The grouping is as follows:

[0069] (1) Control group: fed only the basal diet.

[0070] (2) Experimental group (HTSTCM): 2.667g of the natural plant feed additive prepared in Example 1 was added to every 1kg of basic diet, stirred and mixed evenly, and used for broiler feeding experiment.

[0071] (3) Experimental group (LTSTCM): 1.334g of the natural plant feed additive prepared in Example 2 was added to every 1kg of basic diet, stirred and mixed evenly, and used for broiler feeding experiment.

[0072] (4) Experimental group (HTS): 2.000g of tea saponin extract prepared in Example 2 was added to every 1kg of basic diet, stirred and mixed evenly, and used for broiler feeding experiment.

[0073] (5) Experimental group (LTS): 1.000g of tea saponin extract prepared in Example 2 was added to every 1kg of basic diet, stirred and mixed evenly, and used for broiler feeding experiment.

[0074] (6) Experimental group (TCM): 0.667g of the kudzu root and scutellaria decoction extract prepared in Example 2 was added to every 1kg of basic diet, stirred and mixed evenly, and used for broiler feeding experiment.

[0075] 1.2 Measurement methods for various indicators

[0076] (1) Impact on broiler production performance

[0077] The initial weight of chicks was determined on day 4 before grouping. Weights were taken on days 24 and 43, with the chicks fasted for 12 hours prior to weighing. Average daily weight gain, average daily feed intake, and feed conversion ratio were calculated for each group during experimental period 1 (4–24 days), experimental period 2 (25–43 days), and the entire period (4–43 days). Feed was weighed and daily feed intake was recorded before each feeding of the broiler chickens.

[0078] (2) Morphological observation of broiler intestines (jejunum, ileum, duodenum)

[0079] At the end of 24 and 43 days, one broiler chicken was selected from each replicate. After slaughter, 1 cm of duodenum, jejunum, and ileum were harvested. After carefully removing the intestinal chyme, the samples were rinsed with physiological saline and fixed in 4% paraformaldehyde solution. The fixed tissue samples underwent rinsing, dehydration, clearing, paraffin embedding, embedding, trimming, sectioning, and baking. After routine hematoxylin-eosin (HE) staining, the samples were prepared under a Moticam 3000 optical microscope at 100x magnification to observe the villus height and crypt depth of each tissue. Five intact and well-spread villi were selected from each section, and their villus height, crypt depth, and intestinal wall thickness were measured using ImageJ software. The villus height / crypt depth ratio was calculated, and the average value was taken as the result.

[0080] (3) Effects on the expression levels of ileal barrier genes and proteins in broilers

[0081] At the end of day 43, 100 mg of ileum samples were excised for total RNA extraction, strictly following the instructions. β-actin was used as an internal reference gene, and the levels of occlusive small cyclic 1 (ZO-1), occlusive protein 1 (Claudin 1), and occlusive protein (Occludin) were measured. -ΔΔct The method calculates the relative expression level of the target gene.

[0082] Immunohistochemistry (IHC) is a technique that uses antigen-antibody reactions to detect specific proteins in tissues or cells. Three broiler ileum samples were randomly selected for each group. The tissue samples were fixed with a fixative such as 4% paraformaldehyde to prevent protein degradation. The fixed tissues were dehydrated, embedded in paraffin, and cut into 4-6 micrometer thick sections. After dewaxing and rehydration, antigen retrieval, blocking of endogenous peroxidase, blocking, incubation with primary antibody, incubation with secondary antibody, staining, counterstaining, and mounting, the results were analyzed. The specific procedures were performed by Excellence Biotechnology Co., Ltd.

[0083] Table 1. Primers for intestinal barrier genes

[0084]

[0085] (4) Serum antioxidant capacity assay

[0086] After the feeding trial, at the end of days 24 and 42, six chickens were randomly selected from each replicate. After being given water and fasted for 12 hours, blood was collected from the wing vein. After standing for 2 hours, the blood was centrifuged at 3500 rpm for 15 minutes to prepare serum for the determination of serum antioxidant indicators. The prepared serum was immediately aliquoted and temporarily stored in ice boxes, and then transferred to a -80℃ freezer for storage. The levels of catalase (CAT), malondialdehyde (MDA), total antioxidant capacity (T-AOC), cholesterol (TC), superoxide dismutase (SOD), and glutathione peroxidase (GSH-px) were all measured using a kit and ELISA reader. The kit was purchased from Grace Biotech.

[0087] (5) High-throughput sequencing of gut microbiota

[0088] At the end of day 43, six broiler chickens were selected from each group, and samples of cecal contents were collected and preserved. High-throughput sequencing was then performed on the 16S rDNA V3+V4 regions of these samples. Samples that passed the sequencing were used to construct libraries, and cluster preparation and sequencing were performed using these libraries. Data obtained from the machine were used to perform corresponding bioinformatics analyses. The construction of the high-throughput sequencing libraries was handled by the sequencing company. Based on 97% sequence similarity, these sequences were clustered into operational taxonomic units (OTUs), and the sequences of each cluster and its representative sequence (i.e., OTU) were obtained. Downstream analysis was then performed to calculate the sequence and abundance of each OTU. Database comparisons were conducted, annotation results were filtered, and then sample species complexity and inter-group species differences were analyzed.

[0089] 1.3 Data Processing and Analysis

[0090] Experimental data were analyzed using GraphPad and IBM SPSS 26.0 software for one-way ANOVA, and Duncan's method was used for multiple comparisons. Results are expressed as mean ± standard deviation. A p-value < 0.05 was considered statistically significant.

[0091] 1.4 Results and Analysis

[0092] 1.4.1 Effects of different dosages of the additive of the present invention on the growth performance of broiler chickens

[0093] Depend on Figure 1 It was found that there was no significant difference in body weight among the groups at 4 days. At 24 days, the broiler weight of the TCM group (571.72g) was higher than that of the Control group (540.96g) (p<0.05). At 43 days, the broiler weight of the LTSTCM group (1296.56g) and the TCM group (1285.87g) was significantly higher than that of the Control group (1186.97g) (p<0.01), demonstrating that the addition of low-dose tea saponin compound and high-dose traditional Chinese medicine can significantly increase the body weight of white-feathered broilers at the end of the experiment. At 43 days, the body weight of the HTS group (1104.2g) decreased compared with the Control group (1186.97g), which may be due to the reduced palatability of high-dose tea saponin intake and the resulting decrease in feed intake.

[0094] In the early stage of the experiment (4–24 days), the LTSTCM group (36.80 g / d) showed a significantly lower average daily feed intake compared to the Control group (38.48 g / d) (p < 0.01). In the later stage of the experiment (25–43 days), the HTS group (81.03 g / d) showed a significantly lower average daily feed intake compared to the Control group (89.85 g / d) (p < 0.05). Throughout the entire experiment (4–43 days), the HTS group (58.26 g / d) showed a significantly lower average daily feed intake compared to the Control group (62.88 g / d) (p < 0.05). Other groups showed slight decreases, but these were not significant.

[0095] In the early stage of the experiment (4-24 days), the TCM group (24.72) showed a significant increase in average daily weight gain compared to the Control group (23.30) (p<0.05). In the later stage of the experiment (25-43 days), both the TCM group (34.85) and the LTSCM group (36.59) showed an increase in average daily weight gain compared to the Control group (31.41) (p<0.05), but the LTSCM group showed a highly significant effect (p<0.01). Throughout the entire experiment (4-43 days), both the LTSCM group (29.89) and the TCM group (29.53) showed a highly significant increase in average daily weight gain (p<0.01). The HTS group (25.07) showed a decrease in average daily weight gain compared to the Control group (27.15), which may be because long-term intake of high doses of tea saponins may have a weight-loss effect. Tea saponins may reduce weight by inhibiting fat absorption and regulating lipid metabolism. The average daily weight gain of other groups increased slightly, but there was no significant difference.

[0096] In the early stage of the experiment (4-24 days), the feed conversion ratio (FCR) of both the LTSTCM and TCM groups was significantly lower than that of the Control group (p < 0.05), but the FCR of the LTSTCM group was even lower. In the later stage of the experiment (25-43 days), both the TCM and LTSTCM groups showed a lower FCR compared to the Control group (p < 0.05). Throughout the entire experiment (4-43 days), the FCR of the LTSTCM group was 2.01, and that of the TCM group was 2.04. In conclusion, the addition of the LTSTCM feed additive significantly increased the final weight and average daily weight gain of broiler chickens from day 4 to day 43, reduced the FCR, and decreased the average daily feed intake, but the effect was not significant.

[0097] 1.4.2 Effects of different dosages of the additive of the present invention on the intestinal morphology of 24-day-old white-feathered broiler chickens

[0098] H&E staining of the duodenum was performed at 24 days, such as... Figure 2As shown, the villus height in each group was significantly higher than that in the Control group (p < 0.05). In the duodenum, the villus height in the HTTCM and LTSCM groups was extremely significantly higher than that in the Control group (1.124) (p < 0.01), with the villus height in the LTSCM group being 1.465 and that in the HTTCM group being 1.389. The crypt depth in the HTTCM group (0.235) and HTS group (0.252) was significantly higher than that in the Control group (0.194) (p < 0.05). There were no significant differences among the other groups. The LTSCM group (7.903) and LTS group (7.932) showed significantly higher villus height to crypt depth ratios compared to the Control group (5.892) (p < 0.01). Except for the HTTCM group, which showed no significant change in intestinal wall thickness compared to the Control group, all other groups showed extremely significant increases in intestinal wall thickness (p < 0.01). Specific H&E stained sections are shown in the figure. Figure 2 As shown.

[0099] H&E staining of the jejunum was performed at 24 days, such as... Figure 3 As shown, except for the TCM group, the villus height in all other groups was significantly higher than that in the Control group (p < 0.05). The villus height in the jejunal LTS / LTCM and LTS groups was extremely significantly higher than that in the Control group (0.920) (p < 0.01). The villus height in the LTS / LTCM group was 1.343, and in the LTS group it was 1.294. The crypt depth in the LTS / LTCM group (0.199) and TCM group (0.197) was significantly higher than that in the Control group (0.166) (p < 0.05). 0.05), with no significant differences among the other groups. The LTSTCM group (6.858) and LTS group (7.038) significantly increased the villus height to crypt depth ratio compared to the Control group (5.605) (p < 0.05). Except for the HTTSTCM group, which showed no significant change in intestinal wall thickness compared to the Control group (0.163), all other groups significantly increased intestinal wall thickness (p < 0.05), with the LTSTCM group (0.205) showing a highly significant effect (p < 0.01). Specific H&E stained sections are shown in the image. Figure 3 As shown.

[0100] H&E staining of the ileum was performed at 24 days, such as... Figure 4As shown, the villus height in the TCM group (0.948) was significantly higher than that in the Control group (0.825) (p < 0.01). The crypt depth in the ileum HTSTCM group (0.1507), LTSTCM group (0.1518), and HTS group (0.1423) was significantly lower than that in the Control group (0.1782) (p < 0.05). There were no significant differences among the other groups. The villus height in the LTSTCM group (6.175) and TCM group (5.6) was significantly lower than that in the Control group (0.1782). 90) Compared with the Control group (4.685), all groups significantly increased the ratio of villus height to crypt depth (p < 0.05), with the LTSTCM group showing a more significant effect (p < 0.01). Except for the HTSTCM group, which showed no significant change in intestinal wall thickness compared with the Control group (0.175), all other groups significantly increased intestinal wall thickness (p < 0.05), with the LTSTCM group (0.246) showing a highly significant effect (p < 0.01). Specific H&E stained sections are shown in the figure. Figure 4 As shown.

[0101] 1.4.3 Effects of different dosages of the additive of the present invention on the intestinal morphology of 43-day-old white-feathered broiler chickens

[0102] H&E staining of the duodenum was performed at 43 days, such as... Figure 5 As shown, there was no significant change in villus height in each group at 43 days. The crypt depth decreased slightly in the LTSTCM group (0.257) compared to the Control group (0.330), but the difference was not significant. The LTSTCM group could increase the ratio of duodenal villus height to crypt depth, with a ratio of 5.782 in the LTSTCM group and 4.509 in the Control group. There was no significant difference in intestinal wall thickness among the groups at 43 days. The specific H&E stained sections are shown in the figure.

[0103] H&E staining of the jejunum was performed at 43 days, such as... Figure 6 As shown, the villus height in the HTS group (1.672), LTS group (1.622), and TCM group (1.578) was significantly higher than that in the Control group (1.363) (p < 0.01), while there were no significant differences among the other groups. The crypt depth in the HTS group (0.415) was significantly higher than that in the Control group (0.293) (p < 0.01), while that in the LTS group (0.232) was lower. There were no significant differences among the other groups. The LTS group (7.094) significantly increased the villus height to crypt depth ratio compared to the Control group (4.745) (p < 0.01), and the HTS group (5.904) also significantly increased the jejunal villus height to crypt depth ratio. As for intestinal wall thickness, there were no significant differences among the groups. The specific H&E stained sections are shown in the figure.

[0104] H&E staining of the ileum was performed at 43 days, such as... Figure 7 As shown, the villus height in the LTSTCM group (1.237) was significantly higher than that in the Control group (0.918) (p < 0.01), with no significant differences among other groups. The crypt depth in the ileum was significantly lower in the LTSTCM group (0.162) than in the Control group (0.199) (p < 0.05), with no significant differences among other groups. The LTSTCM group (7.684) significantly increased the villus height to crypt depth ratio compared to the Control group (4.741) (p < 0.01). The ileum wall thickness was significantly increased in the TCM group (0.378) compared to the Control group (0.276), with no significant changes among other groups. The specific H&E stained sections are shown in the figure.

[0105] At 43 days, the LTSTCM group significantly increased the villus height of the duodenum, jejunum, and ileum, while the other groups showed no significant difference in increasing the villus height of the duodenum. The effects of the groups on intestinal wall thickness at 43 days were not significantly different.

[0106] 1.4.4 Effects of different dosages of the additive of the present invention on the ileum intestinal barrier of 43-day-old white-feathered broilers

[0107] Genes related to the intestinal barrier in the ileum were measured, such as... Figure 8 As shown, at 43 days, the expression level of ZO-1 was slightly increased in all groups except the HTSTCM group, with higher expression levels in the LTSTCM group (1.328) and the LTS group (1.205). Compared with the Control group (0.954), the expression level of CLaudin-1 in the LTSTCM group (1.388) was also slightly increased, but not significantly. There was no significant difference in the expression level of Occludin among the groups.

[0108] Three broiler chickens were randomly selected for ileal immunohistochemical analysis, and the results are as follows: Figure 9 As shown, the expression level of ZO-1 protein was significantly increased in the LTSTCM group (p < 0.05). The expression level of Claudin-1 protein was highest in the LTSTCM group, but it was higher than that in the Control group, although the effect was not significant (p > 0.05). The HTSTCM group showed the second highest expression level. The expression levels of Occludin protein were better in the LTSTCM and HTS groups, but not significantly (p > 0.05). Specific immunohistochemical results are shown below. Figure 10-12 As shown.

[0109] 1.4.5 Effect of different amounts of the additive of the present invention on the antioxidant capacity of broiler serum

[0110] Depend on Figure 13It is known that T-AOC refers to the total antioxidant level, composed of various antioxidant substances and antioxidant enzymes, that protects cells and the body from oxidative stress damage caused by reactive oxygen species. Therefore, T-AOC can be used to evaluate the antioxidant capacity of bioactive substances. Cell membranes are composed of lipids. Free radicals generated during animal metabolism can bind to unsaturated fatty acids on the cell membrane, affecting its biological function. The final product of lipid peroxidation is MDA, so its content can, to some extent, reflect the oxidative state of cells.

[0111] For T-AOC, there were no significant differences among the groups at 24 days. At 43 days, the LTSTCM group significantly improved serum total antioxidant capacity (p<0.05), with 0.688 compared to the Control group (0.422). There were no differences among the other groups.

[0112] Regarding total cholesterol (TC), at 24 days, the LTSTC group (2.325) and LTS group (2.415) significantly reduced serum cholesterol levels compared to the Control group (3.415) (p < 0.05), with the LTSTC group showing a better effect (p < 0.01). At 43 days, the LTSTC group showed a slight decrease in TC levels compared to the Control group, from 3.111 to 2.787, but this was not significant, and there were no significant differences among the other groups.

[0113] Regarding MDA, at 24 days, both the LTS and LTS groups reduced serum malondialdehyde (MDA) levels: 0.409 in the Control group, 0.274 in the LTS group, and 0.307 in the LTS group. At 43 days, the LTS group significantly reduced serum MDA levels (p < 0.05): 0.178 in the LTS group, 0.215 in the LTS group, and 0.355 in the Control group.

[0114] For CAT, at 24 days, the LTSTCM group (38.408) significantly increased the serum catalase content compared with the Control group (20.331), with a significant effect (p<0.05). At 43 days, the serum CAT content of each group increased compared with the Control group (11.056), with the LTSTCM group (25.216) showing the best effect, but the results were not statistically significant.

[0115] Regarding SOD, at 24 days, the LTSTCM group (31.886) and HTS group (30.647) showed significantly higher serum superoxide dismutase levels compared to the Control group (19.074) (p < 0.05), while there were no significant differences among the other groups. At 43 days, the LTSTCM group (19.259) showed significantly higher SOD activity compared to the Control group (10.037) (p < 0.01), while SOD activity in the other groups showed slight increases, but no significant differences.

[0116] Regarding glutathione peroxidase, at 24 days, the LTSTCM group (450.216) showed a highly significant increase in activity compared to the Control group (225.826) (p < 0.01), while the LTS group (413.98) and TCM group (400.306) showed significantly increased activity values ​​(p < 0.05). At 43 days, the LTSTCM group (508.057) showed a highly significant increase in activity compared to the Control group (200.803) (p < 0.01), with no significant changes among other groups. In conclusion, the LTSTCM group can improve serum total antioxidant capacity, reduce malondialdehyde content, increase catalase activity, and reduce cholesterol content, which can alleviate oxidative stress in chickens to some extent.

[0117] 1.4.6 Effects of different dosages of the additive of the present invention on the intestinal flora of broiler chickens

[0118] Bioinformatics analysis showed that during the 43-day experimental period, the LTSTCM group and the Control group exhibited significant differences in gut microbiota α-diversity. Figure 14 As shown, the Simpson diversity index was significantly lower in the LTSTCM group (0.37) than in the Control group (0.42) (P < 0.05), while the Shannon diversity index showed a statistically significant upward trend (LTSTCM group: 1.27; Control group: 1.07; P < 0.05). This indicates that the addition of LTSTCM may enhance the biodiversity level of the cecal microbiota by regulating the microbial community structure. Notably, the Chao1 index, a microbial richness indicator, was 11.2% higher in the LTSTCM group (27.75) than in the Control group (23.50) (P > 0.05). Although this difference was not statistically significant, it was still biologically significant.

[0119] A systematic review based on β-diversity analysis revealed significant heterogeneity in the microbial communities between the different treatment groups and the control group. For example... Figure 15As shown, the gut microbiota structure was analyzed using multidimensional statistical methods (principal component analysis (PCA), non-metric multidimensional scaling (NMDS), and principal coordinate analysis (PCOA). The results revealed characteristic distribution patterns in the β-diversity space for each group of samples. Principal component analysis showed that while there was limited spatial overlap between the treatment groups, most areas did not overlap. These results indicate that dietary supplementation with the LTSTCM complex can improve gut microbiota diversity.

[0120] Phylloroplast-level microbial community structure analysis revealed that Bacteroides, Firmicutes, and Actinobacteriota constituted the core microbiota in the cecal flora of 43-day-old broiler chickens. No statistically significant differences were found between the groups and the Control group in phylum-level microbial community composition (P > 0.05), indicating that feed formulation has a conservative influence on macroscopic microbial community structure. Hierarchical clustering heatmap analysis revealed that the relative abundance of Firmicutes in the LTSTCM group (35.63) was lower than that in the control group (43.94) (P > 0.05), while no significant gradient change in Bacteroides was observed among the groups (P > 0.05).

[0121] It is noteworthy that specific functional phyla exhibited differentiated regulatory trends. Verrucomicrobiota, a key bacterial group for mucus layer colonization, showed increased relative abundance in the HTS, LTSCM, and TCM groups compared to the control group, and this phylum was positively correlated with intestinal barrier integrity. Elusimicrobiota, a functional bacterium for fiber degradation, showed increased abundance in both the LTS and LTSCM groups.

[0122] Based on genus-level phylogenetic analysis, the core bacterial genera in the 43-day-old samples included Bacteroides, Ruminococcus, Faecalibacterium, Lactobacillus gasseri, and Clostridium. Cluster heatmap analysis showed that there were differences in the abundance of key functional bacterial genera between the treatment groups and the control group. The relative abundance of Bacteroides was significantly lower in the LTSTCM group (28.15) and HTS group (30.78) than in the Control group (37.09) (P<0.05). Lactobacillus gingivalis, which has probiotic properties, showed significant enrichment in the LTSTCM group (8.21), which was 2.7 times higher than that in the Control group (3.06). The abundance of Femtobacterium in the HTS group (12.03) was upregulated by 43.9% compared with that in the Control group (8.35) (P<0.05), and this genus was positively correlated with butyrate synthesis capacity.

[0123] The LTSTCM group may have significantly optimized the gut microbiota balance through a bidirectional regulatory mechanism, with a lower Firmicutes / Bacteroidetes ratio compared to the Control group. This ratio change was negatively correlated with host energy metabolism efficiency. Predicting pathogenic species phenotypes can provide a better understanding of the relationship between microorganisms and disease. As shown in the figure, the lower the vertical axis, the fewer pathogenic species. At 43 days, the LTSTCM group had the lowest pathogenicity, followed by the LTS group. This indicates that compared to the Control group, there were fewer pathogenic bacteria, and the broiler gut was healthier.

[0124] In summary, the above results indicate that dietary supplementation with LTSTCM can significantly improve the growth performance of broilers, increase intestinal villus height and vitamin C / C ratio, enhance intestinal barrier function, and improve gut microbiota status.

[0125] The embodiments of this application have been described above with reference to the accompanying drawings. Specific examples have been used to illustrate the principles and implementation methods of this application. The description of the above embodiments is only for the purpose of helping to understand the core ideas of this application. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A natural plant feed additive to replace antibiotics in broiler chicken diets, characterized in that, The natural plant feed additive includes tea saponin extract and kudzu root and scutellaria decoction extract; the mass ratio of tea saponin extract to kudzu root and scutellaria decoction extract is 3~9:1; the kudzu root and scutellaria decoction extract is an extract of kudzu root, scutellaria, coptis and prepared licorice. The extract of Ge Gen Qin Lian Tang was obtained by the following extraction method: Kudzu root, Scutellaria baicalensis, Coptis chinensis and prepared licorice root were pulverized and mixed. Then, ultrasonic water extraction was carried out at a material-to-liquid ratio of 1:5~7 and a temperature of 50 ℃~60 ℃. After filtration, water extract and residue were obtained. The dregs were subjected to ultrasonic alcohol extraction at a material-to-liquid ratio of 1:3 to 5, and then filtered to obtain an alcohol extract. The aqueous extract and alcohol extract were concentrated and dried to obtain aqueous extract and alcohol extract, respectively. Finally, the aqueous extract and alcohol extract were mixed to obtain Ge Gen Qin Lian Tang extract. The mass ratio of kudzu root, scutellaria root, coptis root, and prepared licorice root is 4.5~5.5:1.6~2.0:1.6~2.0:1.0~1.

5.

2. The natural plant feed supplement according to claim 1, characterized in that, The mass ratio of water extract to alcohol extract in the Ge Gen Qin Lian Tang extract is 40%~50%: 60%~50%.

3. The natural plant feed supplement according to claim 1, characterized in that, The tea saponin extract was obtained by the following extraction method: Tea seed cake was pulverized to a particle size of 150μm~250μm, dried, and subjected to ultrasonic extraction with ethanol or methanol as the extraction solvent at a material-to-liquid ratio of 1:4~8 and a temperature of 30℃. The extract was then concentrated and dried to obtain tea saponin extract.

4. Natural plant feed additive according to any of claims 1 to 3, characterized in that Both ultrasonic water extraction and ultrasonic alcohol extraction were performed by alternating between focused ultrasound and divergent ultrasound. Each focused ultrasound session lasted 10 to 15 seconds, and each divergent ultrasound session lasted 5 to 10 seconds. The focused extraction power was 1200 W to 3000 W, and the divergent extraction power was 300 W to 1000 W.

5. The natural plant feed supplement according to claim 4, characterized in that, The extraction time for both ultrasonic water extraction and ultrasonic alcohol extraction was 40 min to 60 min.

6. The natural plant feed supplement according to claim 3, characterized in that, After concentration, the product is dried by spray drying, with specific conditions of 120°C air intake and 85°C air outlet.

7. A method of using natural plant feed additive to replace antibiotics in the diet of replacement broiler chickens according to any one of claims 1-6, characterized in that, Add 1.3g to 3.0g of the natural plant feed additive to every 1kg of broiler feed.

8. The use of the natural plant feed additive according to any one of claims 1-6 in the preparation of feed to improve the intestinal health of broilers.

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