A feed additive for improving the high-altitude oxygen tolerance of Tibetan chickens, a preparation method thereof and application thereof

Abstract

This invention discloses a feed additive for improving the tolerance of Tibetan chickens to low oxygen levels at high altitudes, its preparation method, and its application, belonging to the field of feed additive technology. The additive comprises, by weight, 20-22 parts of Atractylodes macrocephala polysaccharide, 8-10 parts of yeast cell wall, 8-10 parts of chlorogenic acid, 4-5 parts of selenium-enriched yeast, 5-7 parts of mannan oligosaccharide, 8-10 parts of Lactobacillus plantarum, 6-8 parts of betaine, 8-10 parts of carnitine, 10-12 parts of dextrin, 8-10 parts of microcrystalline cellulose, and 2-3 parts of montmorillonite powder. The preparation method includes: ultra-finely pulverizing the active ingredients and mixing them at low temperature; adsorbing them using montmorillonite powder as a carrier; and using trehalose to perform low-temperature spray microencapsulation treatment of Lactobacillus plantarum. The feed additive prepared by this invention can significantly enhance the growth performance, survival rate, and physiological adaptability of Tibetan chickens in high-altitude low-oxygen environments.

Description

Technical Field

[0001] This invention belongs to the field of feed additive technology, specifically relating to a feed additive for improving the tolerance of Tibetan chickens to low oxygen levels at high altitudes, its preparation method, and its application. Background Technology

[0002] Tibetan chickens are a unique and high-quality local poultry breed native to the Qinghai-Tibet Plateau of my country. They are characterized by tender meat, unique flavor, and strong adaptability, making them an important part of the local animal husbandry and a key industry for increasing the income of herders. However, the extreme environment of the plateau region, with its high altitude, thin air, and low oxygen partial pressure, poses serious challenges to the growth, development, reproductive performance, and health of Tibetan chickens.

[0003] Existing research indicates that hypoxic environments lead to hypoxic stress responses in Tibetan chickens: on the one hand, decreased blood oxygen saturation, increased respiratory rate, and disordered energy metabolism manifest as stunted growth and reduced feed conversion rate; on the other hand, long-term hypoxia causes myocardial damage, decreased immune function, increased disease incidence, and even death. To improve the high-altitude hypoxia adaptability of Tibetan chickens, existing technologies mainly employ the following measures: first, optimizing the breeding environment, such as building enclosed chicken houses and equipping them with oxygen supply equipment, but these are costly and energy-intensive, making them difficult to promote in remote high-altitude areas; second, adding single anti-stress components to the feed (such as vitamin C, electrolytes, or single probiotics), but these have limited functions and can only alleviate some stress symptoms, failing to fundamentally improve the body's hypoxia tolerance mechanism.

[0004] Although some studies have reported the effect of certain compound additives on improving the stress resistance of poultry, these additives are not specifically designed for Tibetan chickens in the low-oxygen environment of high altitudes, and there is a lack of effective preparation processes to ensure the stability and bioavailability of each active ingredient; in particular, when probiotics coexist with other active ingredients, they are easily inactivated by processing conditions, resulting in unstable product effects.

[0005] Therefore, developing a compound feed additive that can effectively improve the tolerance of Tibetan chickens to low oxygen levels on the plateau has become one of the problems that urgently need to be solved by those skilled in the art. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the present invention provides a feed additive for improving the tolerance of Tibetan chickens to low oxygen at high altitudes, its preparation method, and its application. The present invention comprehensively improves the adaptability of Tibetan chickens to the low oxygen environment at high altitudes through multiple pathways, including antioxidant, immune regulation, intestinal health, and energy metabolism, through the synergistic effect of multiple components. Furthermore, through carrier adsorption and probiotic encapsulation processes, the stability and bioactivity of each active ingredient, especially the probiotics, are ensured.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a feed additive to improve the tolerance of Tibetan chickens to low oxygen at high altitudes. By weight, the feed additive contains the following raw material components: 20-22 parts of Atractylodes macrocephala polysaccharide, 8-10 parts of yeast cell wall, 8-10 parts of chlorogenic acid, 4-5 parts of selenium-enriched yeast, 5-7 parts of mannan oligosaccharide, 8-10 parts of Lactobacillus plantarum, 6-8 parts of betaine, 8-10 parts of carnitine, 10-12 parts of dextrin, 8-10 parts of microcrystalline cellulose, and 2-3 parts of montmorillonite powder.

[0008] Further, by weight, the feed additive comprises the following raw material components: 21 parts of Atractylodes macrocephala polysaccharide, 9 parts of yeast cell wall, 9 parts of chlorogenic acid, 5 parts of selenium-enriched yeast, 6 parts of mannan oligosaccharide, 9 parts of Lactobacillus plantarum, 7 parts of betaine, 9 parts of carnitine, 11 parts of dextrin, 9 parts of microcrystalline cellulose, and 3 parts of montmorillonite powder.

[0009] Furthermore, the *Lactobacillus plantarum* is a freeze-dried powder of *Lactobacillus plantarum* with a viable count of not less than 1 × 10⁻⁶. 9 CFU / g.

[0010] This invention also provides a method for preparing the above-mentioned feed additive to improve the tolerance of Tibetan chickens to low oxygen at high altitudes, comprising the following steps: S1. Raw material preparation and pulverization: Weigh each raw material component according to the weight ratio, and pulverize Atractylodes macrocephala polysaccharide, yeast cell wall, chlorogenic acid, selenium-enriched yeast, mannan oligosaccharide, betaine, and carnitine into ultrafine powders by passing them through a 100-200 mesh sieve; then mix dextrin and microcrystalline cellulose and perform air jet milling, passing them through a 200-300 mesh sieve to obtain carrier mixed powder; S2. Preliminary mixing of active ingredients: Place the ultrafine powders of each raw material obtained in step S1 into a three-dimensional motion mixer, stir and mix at a temperature of 15~25℃ for 20~30min at a speed of 15~25r / min to obtain a mixture of active ingredients; S3. Carrier adsorption: Add montmorillonite powder to the active ingredient mixture obtained in step S2, and continue mixing at 20~30℃ for 10~15 min; then add the carrier mixture powder obtained in step S1, and continue mixing for 15~20 min to obtain the carrier adsorption mixture; S4. Probiotic encapsulation: The freeze-dried powder of Lactobacillus plantarum is mixed with 5-10% (w / v) trehalose aqueous solution and subjected to low-temperature spray encapsulation to form microencapsulated probiotic particles; the microencapsulated probiotic particles are then added to the carrier adsorption mixture obtained in step S3 under aseptic conditions and gently mixed at a low temperature of 10-15℃ for 5-10 min at a speed of 10-15 r / min to obtain a feed additive; S5. Packaging and storage: The feed additives obtained in step S4 are vacuum-packed under nitrogen conditions and stored away from light.

[0011] Further, in step S4, the mass ratio of the freeze-dried Lactobacillus plantarum powder to 5-10% (w / v) trehalose aqueous solution is 1:(1-2).

[0012] Further, in step S4, the process parameters for the low-temperature spray encapsulation treatment are: inlet air temperature 35~45℃, outlet air temperature 20~25℃, and spray pressure 0.1~0.3MPa.

[0013] The present invention also provides the application of the above-mentioned feed additive in the preparation of feed to improve the tolerance of Tibetan chickens to low oxygen at high altitudes, wherein the amount of the feed additive added to the feed is 0.1~0.5wt%, preferably 0.2~0.3wt%.

[0014] The present invention also provides a feed to improve the tolerance of Tibetan chickens to low oxygen at high altitudes, comprising the above-mentioned feed additives, wherein the content of the feed additives is 0.1~0.5wt%, preferably 0.2~0.3wt%.

[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention significantly enhances the adaptability of Tibetan chickens to the high-altitude low-oxygen environment through multiple synergistic effects of immune regulation, antioxidation, intestinal health, and energy metabolism: Atractylodes macrocephala polysaccharide has dual effects of immune regulation and antioxidation, effectively enhancing the immune function of Tibetan chickens and improving the body's resistance; yeast cell wall and mannan oligosaccharide, as high-quality prebiotics, promote the proliferation of beneficial bacteria in the intestine, improve the intestinal barrier function, and enhance the efficiency of nutrient absorption; chlorogenic acid, as a powerful natural antioxidant, effectively removes free radicals and reduces oxidative damage caused by high-altitude low-oxygen stress; selenium-enriched yeast provides a highly bioavailable organic selenium source, significantly enhances the activity of antioxidant enzymes such as glutathione peroxidase, and improves the overall antioxidant capacity of the body; Lactobacillus plantarum regulates the intestinal microecological balance, inhibits the growth of harmful bacteria, enhances nutrient absorption, and at the same time improves the body's immunity through mechanisms such as the gut-lung axis; betaine and carnitine synergistically participate in the energy metabolism process, improve the efficiency of fatty acid β-oxidation and energy utilization efficiency, and effectively alleviate the energy metabolism disorder caused by high-altitude low-oxygen stress.

[0016] (2) The present invention uses low-temperature spray encapsulation technology to microencapsulate Lactobacillus plantarum (inlet air temperature 35~45℃, outlet air temperature 20~25℃), which forms a protective layer while protecting the activity of probiotics, so that they maintain a high number of viable bacteria during subsequent mixing and long-term storage; at the same time, the adsorption properties of montmorillonite powder are used to protect sensitive active ingredients and improve the overall stability of the product; and by first mixing non-probiotic active ingredients and then gently mixing microencapsulated probiotics under low temperature conditions (10~15℃), the process design avoids the adverse effects that may occur if probiotics come into direct contact with other active ingredients. Detailed Implementation

[0017] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0018] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0019] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0020] This invention discloses a feed additive to improve the tolerance of Tibetan chickens to low oxygen at high altitudes. By weight, the feed additive contains the following raw material components: 20-22 parts of Atractylodes macrocephala polysaccharide, 8-10 parts of yeast cell wall, 8-10 parts of chlorogenic acid, 4-5 parts of selenium-enriched yeast, 5-7 parts of mannan oligosaccharide, 8-10 parts of Lactobacillus plantarum, 6-8 parts of betaine, 8-10 parts of carnitine, 10-12 parts of dextrin, 8-10 parts of microcrystalline cellulose, and 2-3 parts of montmorillonite powder.

[0021] The above-mentioned optimal formulation, by weight, comprises the following raw material components: 21 parts Atractylodes macrocephala polysaccharide, 9 parts yeast cell wall, 9 parts chlorogenic acid, 5 parts selenium-enriched yeast, 6 parts mannan oligosaccharide, 9 parts Lactobacillus plantarum, 7 parts betaine, 9 parts carnitine, 11 parts dextrin, 9 parts microcrystalline cellulose, and 3 parts montmorillonite powder. The Lactobacillus plantarum is a freeze-dried powder with a viable count of not less than 1×10⁻⁶. 9 CFU / g.

[0022] This invention also provides a method for preparing the above-mentioned feed additive to improve the high-altitude hypoxia tolerance of Tibetan chickens, comprising the following steps: S1. Raw material preparation and pulverization: Weigh each raw material component according to the weight ratio, and pulverize Atractylodes macrocephala polysaccharide, yeast cell wall, chlorogenic acid, selenium-enriched yeast, mannan oligosaccharide, betaine, and carnitine into ultrafine powders by passing them through a 100-200 mesh sieve; then mix dextrin and microcrystalline cellulose and perform air jet milling, passing them through a 200-300 mesh sieve to obtain carrier mixed powder; S2. Preliminary mixing of active ingredients: Place the ultrafine powders of each raw material obtained in step S1 into a three-dimensional motion mixer, stir and mix at a temperature of 15~25℃ for 20~30min at a speed of 15~25r / min to obtain a mixture of active ingredients; S3. Carrier adsorption: Add montmorillonite powder to the active ingredient mixture obtained in step S2, and continue mixing at 20~30℃ for 10~15 min; then add the carrier mixture powder obtained in step S1, and continue mixing for 15~20 min to obtain the carrier adsorption mixture; S4. Probiotic encapsulation: The freeze-dried powder of Lactobacillus plantarum is mixed with 5-10% (w / v) trehalose aqueous solution and subjected to low-temperature spray encapsulation treatment to form microencapsulated probiotic particles; the microencapsulated probiotic particles are then added to the carrier adsorption mixture obtained in step S3 under aseptic conditions, and gently mixed at a low temperature of 10-15℃ for 5-10 min at a speed of 10-15 r / min to obtain a feed additive; The mass ratio of *Lactobacillus plantarum* freeze-dried powder to 5-10% (w / v) trehalose aqueous solution is 1:(1-2); the process parameters for low-temperature spray encapsulation treatment are: inlet air temperature 35-45℃, outlet air temperature 20-25℃, and spray pressure 0.1-0.3MPa.

[0023] S5. Packaging and storage: The feed additives obtained in step S4 are vacuum-packed under nitrogen conditions and stored away from light.

[0024] The present invention also discloses the application of the above-mentioned feed additive in the preparation of feed to improve the tolerance of Tibetan chickens to low oxygen at high altitudes. The amount of feed additive added to the feed is 0.1~0.5wt%, preferably 0.2~0.3wt%.

[0025] The present invention also discloses a feed for improving the tolerance of Tibetan chickens to low oxygen at high altitudes, comprising the above-mentioned feed additives, wherein the content of the feed additives is 0.1~0.5wt%, preferably 0.2~0.3wt%.

[0026] The technical solution of the present invention will be further described below with reference to specific embodiments: Example 1 (1) Raw material preparation Weigh the following raw material components according to the following weight proportions: Atractylodes macrocephala polysaccharide 21 parts, yeast cell wall 9 parts, chlorogenic acid 9 parts, selenium-enriched yeast 5 parts, mannan oligosaccharide 6 parts, and Lactobacillus plantarum (live count 1.2 × 10⁻⁶). 9 9 parts (CFU / g), 7 parts betaine, 9 parts carnitine, 11 parts dextrin, 9 parts microcrystalline cellulose, and 3 parts montmorillonite powder.

[0027] (2) Preparation method S1. Raw material preparation and pulverization: The above-mentioned Atractylodes macrocephala polysaccharide, yeast cell wall, chlorogenic acid, selenium-enriched yeast, mannan oligosaccharide, betaine and carnitine are pulverized into ultrafine powders by passing them through a 150-mesh sieve; then the dextrin and microcrystalline cellulose are mixed and subjected to air jet pulverization, and passed through a 250-mesh sieve to obtain the carrier mixed powder.

[0028] S2. Preliminary mixing of active ingredients: Place the ultrafine powders of each raw material obtained in step S1 into a three-dimensional motion mixer, stir and mix at 20°C for 25 minutes at a speed of 20 r / min to obtain a mixture of active ingredients.

[0029] S3. Carrier adsorption: Add montmorillonite powder to the active ingredient mixture obtained in step S2 and continue mixing at 25°C for 12 min; then add the carrier mixture powder obtained in step S1 and continue mixing for 18 min to obtain the carrier adsorption mixture.

[0030] S4. Probiotic encapsulation: The freeze-dried powder of Lactobacillus plantarum and 8% (w / v) trehalose aqueous solution were mixed at a mass ratio of 1:1.5. The mixture was then subjected to low-temperature spray encapsulation under the conditions of inlet air temperature of 40℃, outlet air temperature of 22℃ and spray pressure of 0.2MPa to form microencapsulated probiotic particles. The microencapsulated probiotic particles were then added to the carrier adsorption mixture obtained in step S3 under aseptic conditions. The mixture was then gently mixed in a three-dimensional motion mixer at a low temperature of 12℃ for 8 minutes at a speed of 12r / min to obtain a feed additive.

[0031] S5. Packaging and storage: The feed additives obtained in step S4 are vacuum-packed under nitrogen conditions and stored away from light.

[0032] The feed additive prepared in this embodiment was tested, and the viable count of *Lactobacillus plantarum* was 9.8 × 10⁻⁶. 8 The CFU / g content of each component meets the formulation requirements, the product has good flowability, and there is no clumping.

[0033] Example 2 (1) Raw material preparation Weigh the following raw material components according to the following weight proportions: 20 parts of Atractylodes macrocephala polysaccharide, 8 parts of yeast cell wall, 8 parts of chlorogenic acid, 4 parts of selenium-enriched yeast, 5 parts of mannan oligosaccharide, and 1.0 × 10⁻⁶ live bacteria of Lactobacillus plantarum. 9 8 parts (CFU / g), 6 parts betaine, 8 parts carnitine, 10 parts dextrin, 8 parts microcrystalline cellulose, and 2 parts montmorillonite powder.

[0034] (2) Preparation method S1. Raw material preparation and pulverization: The above-mentioned Atractylodes macrocephala polysaccharide, yeast cell wall, chlorogenic acid, selenium-enriched yeast, mannan oligosaccharide, betaine and carnitine are pulverized into ultrafine powders and passed through a 100-mesh sieve to obtain ultrafine powders of each raw material; then, dextrin and microcrystalline cellulose are mixed and subjected to air jet pulverization and passed through a 200-mesh sieve to obtain carrier mixed powder.

[0035] S2. Preliminary mixing of active ingredients: Place the ultrafine powders of each raw material obtained in step S1 into a three-dimensional motion mixer, stir and mix at 15°C for 20 minutes at a speed of 15 r / min to obtain a mixture of active ingredients.

[0036] S3. Carrier adsorption: Add montmorillonite powder to the active ingredient mixture obtained in step S2 and continue mixing at 20°C for 10 min; then add the carrier mixture powder obtained in step S1 and continue mixing for 15 min to obtain the carrier adsorption mixture.

[0037] S4. Probiotic Encapsulation: The freeze-dried powder of Lactobacillus plantarum and 5% (w / v) trehalose aqueous solution were mixed at a mass ratio of 1:1. The mixture was then subjected to low-temperature spray encapsulation under the conditions of inlet air temperature of 35℃, outlet air temperature of 20℃, and spray pressure of 0.1MPa to form microencapsulated probiotic particles. The microencapsulated probiotic particles were then added to the carrier adsorption mixture obtained in step S3 under aseptic conditions. The mixture was then gently mixed in a three-dimensional motion mixer at a low temperature of 10℃ for 5 minutes at a rotation speed of 10r / min to obtain a feed additive.

[0038] S5. Packaging and storage: The feed additives obtained in step S4 are vacuum-packed under nitrogen conditions and stored away from light.

[0039] The feed additive prepared in this embodiment was tested, and the viable count of *Lactobacillus plantarum* was 8.5 × 10⁻⁶. 8 The CFU / g content of each component meets the formulation requirements, the product has good flowability, and there is no clumping.

[0040] Example 3 (1) Raw material preparation Weigh the following raw material components according to the indicated weight proportions: 22 parts Atractylodes macrocephala polysaccharide, 10 parts yeast cell wall, 10 parts chlorogenic acid, 5 parts selenium-enriched yeast, 7 parts mannan oligosaccharide, and 1.5 × 10⁻⁶ live bacteria of Lactobacillus plantarum. 9 10 parts (CFU / g), 8 parts betaine, 10 parts carnitine, 12 parts dextrin, 10 parts microcrystalline cellulose, and 3 parts montmorillonite powder.

[0041] (2) Preparation method S1. Raw material preparation and pulverization: The above-mentioned Atractylodes macrocephala polysaccharide, yeast cell wall, chlorogenic acid, selenium-enriched yeast, mannan oligosaccharide, betaine and carnitine are pulverized into ultrafine powders and passed through a 200-mesh sieve to obtain ultrafine powders of each raw material; then, dextrin and microcrystalline cellulose are mixed and subjected to air jet pulverization and passed through a 300-mesh sieve to obtain carrier mixed powder.

[0042] S2. Preliminary mixing of active ingredients: Place the ultrafine powders of each raw material obtained in step S1 into a three-dimensional motion mixer, stir and mix at 25°C for 30 minutes at a speed of 25 r / min to obtain a mixture of active ingredients.

[0043] S3. Carrier adsorption: Add montmorillonite powder to the active ingredient mixture obtained in step S2 and continue mixing at 30°C for 15 min; then add the carrier mixture powder obtained in step S1 and continue mixing for 20 min to obtain the carrier adsorption mixture.

[0044] S4. Probiotic Encapsulation: The freeze-dried powder of Lactobacillus plantarum and a 10% (w / v) trehalose aqueous solution were mixed at a mass ratio of 1:2. The mixture was then subjected to low-temperature spray encapsulation under the conditions of an inlet air temperature of 45℃, an outlet air temperature of 25℃, and a spray pressure of 0.2MPa to form microencapsulated probiotic particles. The microencapsulated probiotic particles were then added to the carrier adsorption mixture obtained in step S3 under aseptic conditions. The mixture was then gently mixed in a three-dimensional motion mixer at a low temperature of 15℃ for 10 minutes at a rotation speed of 15r / min to obtain a feed additive.

[0045] S5. Packaging and storage: The feed additives obtained in step S4 are vacuum-packed under nitrogen conditions and stored away from light.

[0046] The feed additive prepared in this embodiment was tested, and the viable count of *Lactobacillus plantarum* was 1.1 × 10⁻⁶. 9 The CFU / g content of each component meets the formulation requirements, the product has good flowability, and there is no clumping.

[0047] Example 4 This embodiment verifies the effect of the feed additive of the present invention on improving the growth performance, survival rate, hypoxia tolerance, antioxidant and immune function of Tibetan chickens in high-altitude environments.

[0048] The experiment used 360 healthy 1-day-old male Tibetan chickens with an average birth weight of 34.5 ± 1.2 g. They were randomly divided into 4 groups (control group, experimental groups 1, 2, and 3), with 6 replicates per group and 15 chickens per replicate. The experiment was conducted at a plateau farm at an altitude of 3650 m for 18 weeks.

[0049] Control group: fed with a basal diet; Experimental group 1: basal diet + feed additive prepared in Example 1 (addition amount 0.1wt%). Experimental group 2: basal diet + feed additive prepared in Example 1 (addition amount 0.3wt%); Experimental group 3: basal diet + feed additive prepared in Example 1 (addition amount 0.5wt%); During the experiment, the fasting weight of each replicate chicken was recorded at a fixed time each week to calculate the average daily weight gain, and the feed consumption was recorded to calculate the feed conversion ratio; mortality was observed and recorded daily.

[0050] During week 18 of the experiment, two chickens were randomly selected from each replicate for sampling. Blood was collected via the wing vein, and hemoglobin (g / dL) and red blood cell count (×10⁻⁶) were measured using a hematology analyzer. 12 Blood oxygen saturation (%) was measured using a pulse oximeter. After blood collection, the blood was allowed to stand for 30 min, then centrifuged at 3000 r / min for 10 min to separate the serum, which was then stored at -80℃. Using kits from Nanjing Jiancheng Bioengineering Institute, the activities of superoxide dismutase (SOD, U / mL), glutathione peroxidase (GSH-Px, U / mL), and malondialdehyde (MDA, nmol / mL) in the serum were measured. Subsequently, the experimental chickens were euthanized, the spleen was completely removed and weighed, and the immune organ index (organ weight / body weight × 100%) was calculated. Simultaneously, a portion of liver tissue was prepared into a 10% homogenate, and the catalase (CAT, U / mg prot) activity was measured. Separately, serum was collected, and the levels of immunoglobulins IgG and IgM (g / L) were measured using ELISA. Data were analyzed using SPSS 22.0 software via one-way ANOVA, and Duncan's multiple comparison method was used to test for significance between groups. Results are expressed as mean ± standard deviation.

[0051] The test results are as follows: (1) Regarding growth performance, as shown in Table 1.

[0052] Table 1

[0053] Note: "Average daily weight gain" in the table refers to the average daily weight gain during the period from week 6 to week 18 (i.e., day 43 to day 126, a total of 84 days). As shown in Table 1, in terms of growth performance, experimental groups 1-3 all showed varying degrees of improvement compared to the control group. Among them, experimental group 2 (0.3% supplementation) showed the most significant effect, with its body weight reaching 932.6±28.4g at 18 weeks, an increase of approximately 10.2% compared to the control group (846.6±33.5g); the average daily weight gain was 7.99±0.4g, an increase of approximately 5.8% compared to the control group (7.55±0.6g); and the feed conversion ratio decreased to 4.68±0.09, a decrease of approximately 11.4% compared to the control group (5.28±0.13).

[0054] (2) Regarding mortality rates, see Table 2.

[0055] Table 2

[0056] As shown in Table 2, the overall mortality rate of experimental groups 1-3 was lower than that of the control group (8.5±1.5%). The overall mortality rate of experimental group 2 was the lowest, at 3.8±0.8%, which was about 55.3% lower than that of the control group.

[0057] (3) Regarding high-altitude hypoxia tolerance (18 weeks), as shown in Table 3.

[0058] Table 3

[0059] As can be seen from Table 3, in terms of survival rate and tolerance to high altitude hypoxia, experimental groups 1-3 were better than the control group; the blood oxygen saturation of experimental group 2 (91.8±1.5%) was about 4.6% higher than that of the control group (87.8±2.1%), and the hemoglobin level (14.6±0.8g / dL) was 15.9% higher than that of the control group (12.6±1.0). (4) In terms of antioxidant capacity (18 weeks), as shown in Table 4.

[0060] Table 4

[0061] As shown in Table 4, in terms of antioxidant capacity, experimental groups 1-3 were better than the control group; the serum SOD activity of experimental group 2 (251.3±12.1U / mL) was about 37.0% higher than that of the control group (183.5±13.2U / mL), and the MDA content (5.6±0.6nmol / mL) was about 36.4% lower than that of the control group (8.8±0.8).

[0062] (5) Regarding immune performance (18 weeks), see Table 5.

[0063] Table 5

[0064] As can be seen from Table 5, in terms of immune performance, the serum IgG level of experimental group 2 (11.4±0.7g / L) was about 40.7% higher than that of the control group (8.1±0.7g / L), and the spleen index (0.39±0.03%) was about 44.4% higher than that of the control group (0.27±0.03), showing a strong immune-enhancing effect.

[0065] In summary, the feed additive of this invention can effectively improve various physiological indicators of Tibetan chickens in high-altitude environments within the addition range of 0.1~0.5wt%, with 0.2~0.3wt% being the optimal addition range and 0.3wt% being the optimal addition amount. The additive prepared by this invention, through the synergistic effect of multiple components, significantly improves the growth performance, survival rate, high-altitude hypoxia tolerance, antioxidant capacity, and immune function of Tibetan chickens.

[0066] Comparative Example 1 This comparative example is a feed additive formulated to address the lack of Lactobacillus plantarum.

[0067] The formulation of this comparative feed additive is the same as that in Example 1, except that Lactobacillus plantarum is omitted; specifically, it consists of 21 parts of Atractylodes macrocephala polysaccharide, 9 parts of yeast cell wall, 9 parts of chlorogenic acid, 5 parts of selenium-enriched yeast, 6 parts of mannan oligosaccharide, 7 parts of betaine, 9 parts of carnitine, 11 parts of dextrin, 9 parts of microcrystalline cellulose, and 3 parts of montmorillonite powder.

[0068] The preparation process of this comparative feed additive is the same as that in Example 1, except that step S4, probiotic encapsulation, is omitted.

[0069] The experiment selected 180 healthy 1-day-old male Tibetan chickens, the same as those in Example 4, and randomly divided them into Example 1 group and Comparative Example 1 group, with 6 replicates in each group and 15 chickens in each replicate; they were fed for 18 weeks in a plateau farm at an altitude of 3650 m, with an additive amount of 0.3 wt.

[0070] Example 1 group: fed with basal diet + feed additive prepared in Example 1 (addition amount 0.3wt%). Comparative Example 1: The diet was basal and supplemented with the feed additive prepared in Comparative Example 1 (addition amount 0.3wt%).

[0071] The results of the comparative experiment on the production and physiological indicators of Tibetan chickens in Example 1 and Comparative Example 1 are shown in Table 6.

[0072] Table 6

[0073] As can be seen from Table 6: (i) In terms of growth performance, the body weight of Group 1 at 18 weeks was 932.6±28.4 g, which was significantly higher than that of Group 1 (861.2±30.1 g); the average daily weight gain of Group 1 was 7.99±0.4 g, which was about 17.7% higher than that of Group 1 (6.79±0.5 g); and the feed conversion ratio of Group 1 was 4.68±0.09, which was about 6.0% lower than that of Group 1 (4.98±0.11).

[0074] (ii) Survival rate: The overall mortality rate of Example 1 group was 3.8±0.8%, which was significantly lower than that of Comparative Example 1 group (6.9±0.3%), a reduction of approximately 44.9%.

[0075] (iii) Regarding hypoxia tolerance: The blood oxygen saturation of the Example 1 group was 91.8±1.5%, which was higher than that of the Comparative Example 1 group (87.4±2.0%); the serum IgG level was 11.4±0.7 g / L, which was about 16.3% higher than that of the Comparative Example 1 group (9.8±0.6 g / L).

[0076] The above experiments demonstrate that while the feed additive prepared with *Lactobacillus plantarum* missing in this comparative example still has some effect, its efficacy in promoting growth, reducing mortality, and enhancing hypoxia adaptability is significantly weaker than that of the feed additive prepared in Example 1 of this invention. This indicates that *Lactobacillus plantarum* is the core component for achieving healthy breeding of Tibetan chickens in the plateau region. It forms a synergistic "bacterium-polysaccharide-antioxidant" system with *Atractylodes macrocephala* polysaccharide, mannan oligosaccharide, etc., jointly enhancing the adaptability of Tibetan chickens to the low-oxygen environment of the plateau.

[0077] Comparative Example 2 The formulation of this comparative feed additive is exactly the same as that in Example 1.

[0078] The preparation process of this comparative example is as follows: except for step S4, in which the freeze-dried Lactobacillus plantarum powder is directly added to the carrier adsorption mixture obtained in step S3, and mixed at a low temperature of 12°C for 8 min at a rotation speed of 12 r / min to obtain the feed additive (without microencapsulation treatment), the remaining steps are the same as in Example 1.

[0079] The experiment selected 180 healthy 1-day-old male Tibetan chickens, the same as those in Example 4, and randomly divided them into Example 1 group and Comparative Example 2 group, with 6 replicates in each group and 15 chickens in each replicate; they were fed for 18 weeks in a plateau farm at an altitude of 3650 m, with an additive amount of 0.3 wt.

[0080] Example 1 group: fed with basal diet + feed additive prepared in Example 1 (addition amount 0.3wt%). Comparative Example 2: fed with a basal diet plus the feed additive prepared in Comparative Example 2 (addition amount 0.3wt%).

[0081] The test results are shown in Table 7 (survival rate of Lactobacillus plantarum in feed additives under different processes) and Table 8 (comparative test results of Tibetan chicken production and physiological indicators).

[0082] Table 7

[0083] As can be seen from Table 7, after preparation, the viable bacterial count in Example 1 group was 9.8 × 10⁻⁶. 8CFU / g, comparative group 2 was 9.5×10 8 CFU / g, initially no significant difference; after 30 days of storage, the CFU / g of Example 1 group remained at 8.6 × 10⁻⁶. 8 CFU / g, while the comparative group 2 dropped sharply to 5.2×10⁻⁶. 8 CFU / g; After storage for 60 days: Group 1 of Example 1 had a concentration of 7.9 × 10⁻⁶ CFU / g. 8 CFU / g, compared to only 2.8 × 10⁻⁶ in the two comparative groups. 8 CFU / g; After 90 days of storage: Group 1 of Example 1 had a concentration of 7.2 × 10⁻⁶ CFU / g. 8 CFU / g, compared to only 1.1×10 in the two comparative groups. 8 CFU / g.

[0084] This indicates that unencapsulated probiotics are highly susceptible to inactivation during storage, with an activity loss of approximately 88.4% after 90 days compared to when they were first prepared; while probiotics that have undergone encapsulation processes show a lower activity loss during storage, with an activity loss of approximately 26.5% after 90 days compared to when they were first prepared.

[0085] Table 8

[0086] As can be seen from Table 8, if the microencapsulation process of *Lactobacillus plantarum* is omitted, the number of viable bacteria in the product decreases sharply during storage, resulting in weaker growth performance, survival rate, and immune indicators in Tibetan chickens after feeding compared to the group in Example 1 of this invention. This indicates that the encapsulation technology provided by this invention ensures the stability of probiotic activity.

[0087] Comparative Example 3 The formulation of this comparative feed additive is exactly the same as that in Example 1.

[0088] The preparation process of this comparative example is the same as that of Example 1, except for step S4, which involves "conventional high-temperature spray drying with process parameters of 150°C inlet air temperature, 80°C outlet air temperature, and 0.2MPa spray pressure".

[0089] The experiment selected 180 healthy 1-day-old male Tibetan chickens, the same as those in Example 4, and randomly divided them into Example 1 group and Comparative Example 3 group, with 6 replicates in each group and 15 chickens in each replicate; they were fed for 18 weeks in a plateau farm at an altitude of 3650m, with an additive amount of 0.3wt.

[0090] Example 1 group: fed with basal diet + feed additive prepared in Example 1 (addition amount 0.3wt%). Comparative Example 3: fed with a basal diet plus the feed additive prepared in Comparative Example 3 (addition amount 0.3wt%).

[0091] The test results are shown in Table 9 (survival rate of Lactobacillus plantarum in feed additives under different processes) and Table 10 (comparative test results of Tibetan chicken production and physiological indicators).

[0092] Table 9

[0093] Table 10

[0094] As shown in Tables 9 and 10, the high-temperature encapsulation process used in this comparative example resulted in only 3.3% of the viable bacteria count in Example 1 after preparation, indicating that high temperature severely damaged the probiotic activity. Furthermore, the growth performance, survival rate, and immune indicators of the Tibetan chickens were significantly lower than those in Example 1. This demonstrates that high-temperature treatment severely damages the cell structure of probiotics, causing them to lose their biological activity and fail to perform their intended functions.

[0095] Comparative Example 4 The formulation of this comparative feed additive is exactly the same as that in Example 1.

[0096] The preparation process of this comparative example is as follows: Step S2 is changed to placing all the ultrafine powders of raw materials obtained in step S1 (including Atractylodes macrocephala polysaccharide, yeast cell wall, chlorogenic acid, selenium-enriched yeast, mannan oligosaccharide, betaine, and carnitine), as well as montmorillonite powder, dextrin, and microcrystalline cellulose, into a three-dimensional motion mixer and stirring at 20°C for 35 minutes at a speed of 20 r / min to obtain a mixture. Step S3 is omitted, and carrier adsorption is not performed. The rest is the same as in Example 1.

[0097] The experiment selected 180 healthy 1-day-old male Tibetan chickens, the same as those in Example 4, and randomly divided them into Example 1 group and Comparative Example 4 group, with 6 replicates in each group and 15 chickens in each replicate; they were fed for 18 weeks in a plateau farm at an altitude of 3500 m, with an additive amount of 0.3 wt.

[0098] Example 1 group: fed with basal diet + feed additive prepared in Example 1 (addition amount 0.3wt%). Comparative Example 4: fed with a basal diet plus the feed additive prepared in Comparative Example 4 (addition amount 0.3wt%).

[0099] The test results are shown in Tables 11 and 12.

[0100] Table 11

[0101] Table 12

[0102] As can be seen from Tables 11 and 12, in this comparative example, omitting the carrier adsorption process in step S3 and only dry-mixing all raw materials, despite having the same initial formula, the product agglomeration rate significantly increased, the retention rate of active ingredients (chlorogenic acid and betaine) decreased drastically, and the number of surviving probiotics decreased. Consequently, the growth performance, survival rate, and hypoxia tolerance of Tibetan chickens fed with this product were all inferior to those of Example 1. This demonstrates that the carrier adsorption of the present invention is a necessary technical means to achieve stable coexistence, targeted delivery, and functional synergy of multiple components, which cannot be replaced by conventional mixing processes in this field.

[0103] The above provides a detailed description of a feed additive for improving the high-altitude hypoxia tolerance of Tibetan chickens, its preparation method, and its application. Specific examples have been used to illustrate the principles and implementation methods of this invention. The descriptions of these examples are merely for the purpose of helping to understand the method and core ideas of this invention. It should be noted that those skilled in the art can make various improvements and modifications to this invention without departing from its principles, and these improvements and modifications also fall within the scope of protection of the claims of this invention.

Claims

1. A feed additive for improving the tolerance of Tibetan chickens to low oxygen levels at high altitudes, characterized in that, By weight, the feed additive comprises the following raw material components: 20-22 parts of Atractylodes macrocephala polysaccharide, 8-10 parts of yeast cell wall, 8-10 parts of chlorogenic acid, 4-5 parts of selenium-enriched yeast, 5-7 parts of mannan oligosaccharide, 8-10 parts of Lactobacillus plantarum, 6-8 parts of betaine, 8-10 parts of carnitine, 10-12 parts of dextrin, 8-10 parts of microcrystalline cellulose, and 2-3 parts of montmorillonite powder.

2. The feed additive according to claim 1, characterized in that, By weight, the feed additive contains the following raw material components: 21 parts of Atractylodes macrocephala polysaccharide, 9 parts of yeast cell wall, 9 parts of chlorogenic acid, 5 parts of selenium-enriched yeast, 6 parts of mannan oligosaccharide, 9 parts of Lactobacillus plantarum, 7 parts of betaine, 9 parts of carnitine, 11 parts of dextrin, 9 parts of microcrystalline cellulose, and 3 parts of montmorillonite powder.

3. The feed additive according to claim 1, characterized in that, The *Lactobacillus plantarum* mentioned is a freeze-dried powder of *Lactobacillus plantarum* with a viable count of not less than 1×10⁻⁶. 9 CFU / g.

4. A method for preparing a feed additive for improving the high-altitude hypoxia tolerance of Tibetan chickens as described in any one of claims 1-3, characterized in that, Includes the following steps: S1. Raw material preparation and pulverization: Weigh each raw material component according to the weight ratio, and pulverize Atractylodes macrocephala polysaccharide, yeast cell wall, chlorogenic acid, selenium-enriched yeast, mannan oligosaccharide, betaine, and carnitine into ultrafine powders by passing them through a 100-200 mesh sieve; then mix dextrin and microcrystalline cellulose and perform air jet milling, passing them through a 200-300 mesh sieve to obtain carrier mixed powder; S2. Preliminary mixing of active ingredients: Place the ultrafine powders of each raw material obtained in step S1 into a three-dimensional motion mixer, stir and mix at a temperature of 15~25℃ for 20~30min at a speed of 15~25r / min to obtain a mixture of active ingredients; S3. Carrier adsorption: Add montmorillonite powder to the active ingredient mixture obtained in step S2, and continue mixing at 20~30℃ for 10~15 min; then add the carrier mixture powder obtained in step S1, and continue mixing for 15~20 min to obtain the carrier adsorption mixture; S4. Probiotic encapsulation: The freeze-dried powder of Lactobacillus plantarum is mixed with 5-10% (w / v) trehalose aqueous solution and subjected to low-temperature spray encapsulation treatment to form microencapsulated probiotic particles; the microencapsulated probiotic particles are then added to the carrier adsorption mixture obtained in step S3 under aseptic conditions, and gently mixed at a low temperature of 10-15℃ for 5-10 min at a speed of 10-15 r / min to obtain a feed additive; S5. Packaging and storage: The feed additives obtained in step S4 are vacuum-packed under nitrogen conditions and stored away from light.

5. The preparation method according to claim 4, characterized in that, Step S4, the mass ratio of the freeze-dried Lactobacillus plantarum powder to 5-10% (w / v) trehalose aqueous solution is 1:(1-2).

6. The preparation method according to claim 4 or 5, characterized in that, Step S4, the process parameters for the low-temperature spray encapsulation treatment are: inlet air temperature 35~45℃, outlet air temperature 20~25℃, and spray pressure 0.1~0.3MPa.

7. The use of a feed additive as described in any one of claims 1-3 in the preparation of feed to improve the tolerance of Tibetan chickens to low oxygen at high altitudes.

8. The application according to claim 7, characterized in that, The feed additive is added to the feed at a rate of 0.1~0.5 wt%.

9. A feed for improving the tolerance of Tibetan chickens to low oxygen levels at high altitudes, characterized in that, The feed additive comprises any one of claims 1-3, wherein the content of the feed additive is 0.1-0.5 wt%.

10. The feed according to claim 9, characterized in that, The content of the feed additive is 0.2~0.3wt%.

Images