Composite additive for laying fowl health care and preparation method thereof

Abstract

The invention discloses a laying fowl health-care composite additive and a preparation method thereof, and belongs to the technical field of nutrition and health care for laying hen breeding. The compound additive takes water as a carrier, contains microencapsulated vitamin A, coated vitamin C, micro-emulsified vitamin E and 25-hydroxyvitamin D3 as well as low-temperature coated probiotics and ultrasonic-enzymolysis selenium yeast, and solves the problems of poor stability, low bioavailability, inconvenience in administration and the like of a traditional additive. The preparation method comprises the following steps: microencapsulation / nanoemulsion embedding, directional fermentation and concentration of probiotics, organic selenium ultrasonic-enzymolysis pretreatment, and compounding and blending under the protection of nitrogen. The additive is of a clarified microemulsion type, can significantly improve comprehensive benefits of egg poultry breeding, achieves the goals of high yield, excellent shell, rich selenium and low death and culling, is suitable for large-scale automatic breeding, and has a shelf life of more than or equal to 12 months at 25 DEG C.

Description

Technical Field

[0001] This invention belongs to the technical field of nutritional and health care products for egg-laying hen farming, specifically relating to a compound additive for the health care of egg-laying poultry and its preparation method. Background Technology

[0002] Currently, the egg-laying hen farming industry is rapidly developing towards large-scale and intensive farming. While large-scale farming improves farming efficiency and ensures egg production supply, it also presents egg-laying hens with more complex survival challenges. The combined effects of multiple adverse factors, such as high-production stress, immunosuppression, gut microbiota imbalance, and oxidative stress, lead to frequent problems in egg-laying hen farming, including decreased egg production, poor eggshell quality, increased mortality, and frequent vitamin deficiencies. These issues severely restrict the healthy and sustainable development of the egg-laying hen farming industry and cause significant economic losses to farming enterprises.

[0003] To address the aforementioned issues, traditional egg-laying hen farming often employs nutritional regulation methods, primarily relying on powdered multivitamins, antibiotics, or single probiotic additives. However, these traditional regulatory products have numerous shortcomings in practical application, failing to meet the high-efficiency demands of large-scale, intensive farming. Specific problems include: (1) Fat-soluble vitamins A and E, which are essential for the growth, development and egg production of laying hens, are easily oxidized and become ineffective in aqueous environments, resulting in poor stability. Water-soluble vitamin C has strong degradation characteristics and its activity is easily lost during storage and use, ultimately leading to these vitamins not being able to fully play their role and their bioavailability being greatly reduced.

[0004] (2) 25-hydroxyvitamin D3, as the active form of vitamin D, should have better physiological efficacy. However, under the traditional powder addition method, its absorption effect is significantly affected by the fat content in the feed, resulting in unstable absorption process. It cannot ensure the effective intake of this active vitamin by laying hens, and the absorption efficiency is limited, making it difficult to give full play to its role in regulating calcium and phosphorus metabolism and improving eggshell quality.

[0005] (3) Commonly used probiotics such as Clostridium butyricum, Bacillus licheniformis, and Bacillus subtilis are easily inactivated in powdered preparations due to environmental factors, resulting in low probiotic colonization rates. At the same time, when these probiotics are administered through drinking water in large-scale farming, they are also affected by factors such as water pH and disinfectant residues, further reducing their colonization ability in the intestines of laying hens. They cannot effectively regulate the balance of intestinal flora and it is difficult to achieve the ideal intestinal health care effect.

[0006] (4) Organic selenium, such as yeast selenium, is an important nutrient for enhancing the antioxidant capacity of laying hens and improving egg quality. However, in traditional powdered additives, organic selenium is prone to antagonistic reactions with trace elements in feed, which significantly reduces its deposition efficiency in laying hens, thus reducing the utilization rate of organic selenium and failing to fully exert its physiological functions of antioxidant and enhancing the body's immunity.

[0007] (5) Traditional powdered additives need to be used by mixing with feed. For large-scale chicken farms, this operation method is labor-intensive and consumes a lot of manpower and resources. At the same time, uneven mixing is likely to occur during the mixing process, resulting in different intakes of different laying hens, making it difficult to achieve precise drug administration. In fact, the local dosage may be too high or too low, affecting the control effect and increasing the stress response of laying hens.

[0008] In summary, existing traditional nutritional regulation methods suffer from numerous drawbacks, including poor stability, low bioavailability, inconvenient administration, insufficient precision, limited functionality, and inability to achieve synergistic effects. These limitations make it difficult to effectively address the multiple challenges faced by laying hens in large-scale, intensive farming models. Therefore, developing a liquid health product that is highly stable, bioavailable, conveniently administered via drinking water, and capable of synergistic effects from multiple functional components such as vitamins, probiotics, and organic selenium to comprehensively enhance the immunity of laying hens, improve eggshell quality, and extend the peak laying period has become an urgent need in the current laying hen farming industry. Summary of the Invention

[0009] Based on the above technical background, the main objective of this invention is to provide a compound additive for the health care of poultry and its preparation method, so as to overcome the shortcomings of the prior art.

[0010] To achieve the aforementioned objectives, the technical solution adopted by this invention includes: The first aspect of the present invention is to provide a compound additive for the health care of poultry, the compound additive comprising vitamin A, vitamin C, vitamin E, 25-hydroxyvitamin D3, Clostridium butyricum, Bacillus licheniformis, organic selenium, stabilizer, pH buffer system and water.

[0011] The amounts of each ingredient in the 1 L composite additive are as follows: Vitamin A 500,000–1,000,000 IU, Vitamin C 5–10 g, Vitamin E 2–5 g, 25-hydroxyvitamin D3 2.5–4 g, and butyric acid bacteria ≥1×10 9 CFU / mL (with viable bacterial count ≥ 1 billion), Bacillus licheniformis ≥ 5 × 10⁻⁶ 8 CFU / mL (with viable bacteria count ≥ 500 million), organic selenium 0.5–1.0 g, stabilizer 10–15 g, the amount of pH buffer system added should be sufficient to adjust the pH of the compound additive to 6.0–6.5, the remainder being water.

[0012] Preferably, the amounts of each raw material in the 1 L composite additive are: Vitamin A 700,000 IU, Vitamin C 7 g, Vitamin E 3 g, 25-hydroxyvitamin D3 3 g, and Butyric acid bacteria ≥1×10 9 CFU / mL (with viable bacterial count ≥ 1 billion), Bacillus licheniformis ≥ 5 × 10⁻⁶ 8 CFU / mL (with viable bacteria count ≥ 500 million), 0.8 g of organic selenium, 12 g of stabilizer, the amount of pH buffer added should be sufficient to adjust the pH of the compound additive to 6.0-6.5, the remainder is water.

[0013] The vitamin A mentioned is water-soluble microencapsulated vitamin A. Vitamin A has antioxidant properties and can also maintain the integrity of mucous membranes.

[0014] The vitamin C mentioned above has anti-stress and iron-absorbing properties.

[0015] The vitamin E mentioned above has antioxidant and cell membrane protective effects.

[0016] The 25-hydroxyvitamin D3 can promote calcium and phosphorus absorption and improve eggshell quality.

[0017] The butyric acid bacteria can produce butyric acid, repair the intestinal mucosa, and inhibit Clostridium.

[0018] The Bacillus licheniformis can promote the colonization of anaerobic bacteria and enzyme production.

[0019] The organic selenium is preferably yeast selenium, with Se≥0.2%. Organic selenium has the effects of anti-oxidation, enhancing selenium deposition, and strengthening immunity.

[0020] The pH buffer solution is sodium citrate + phosphate. First, prepare 0.1 mol / L citric acid solution and 0.2 mol / L Na2HPO4 solution separately, and then adjust the corresponding volume ratio according to the required pH.

[0021] The stabilizer is selected from one or more of glycerol, sorbitol, and BHA (butylated carboxyanisole).

[0022] Preferably, the stabilizer is a mixture of glycerol and sorbitol, wherein the mass ratio of glycerol to sorbitol is (2-5):1. The stabilizer has moisturizing, anti-crystallization, and cell-protecting functions.

[0023] More preferably, the mass ratio of glycerol to sorbitol is 4:1.

[0024] The pH buffer system can protect the activity of vitamins and probiotics.

[0025] All vitamins used in the above-mentioned raw materials of this invention are produced using water-soluble microencapsulation or nanoemulsification technology, and probiotics are produced using low-temperature coating + storage-resistant liquid carrier technology to ensure that their shelf life is ≥12 months at 25°C.

[0026] The composite additive described in this invention achieves synergistic effects across multiple components: the vitamin oxidation half-life is extended to ≥300 days, the bioavailability of 25-hydroxyvitamin D3 reaches 188%, the 12-month survival rate of probiotics is ≥85%, and the release of selenomethionine from yeast is increased by 42%. After application, the eggshell thickness of laying hens increases by 0.04 mm, the breakage rate decreases by 35%, the egg production rate increases by 5.3 percentage points, the mortality rate decreases to 0.25%, and the selenium content of the egg yolk meets the standard for selenium-enriched eggs. Furthermore, the product is non-toxic, has no withdrawal period, and meets the requirements of green farming. A second aspect of the present invention is to provide a method for preparing the composite additive described in the first aspect of the present invention, the method comprising the following steps: Step 1: Microencapsulate vitamin A to make it water-soluble, and microemulse vitamin E and 25-OH-D3 to make a water-soluble vitamin. Step 2: Spray-coat vitamin C with ethyl cellulose and then vacuum dry to obtain ethyl cellulose-coated vitamin C; Step 3: Ferment Clostridium butyricum and Bacillus licheniformis, collect the bacterial cells by centrifugation, and prepare a concentrated bacterial suspension using glycerol-sorbitol composite stabilizer; Step 4: The organic selenium is subjected to ultrasonic crushing and enzymatic hydrolysis in sequence to obtain the organic selenium pretreated product; Step 5: Under nitrogen protection, water-soluble vitamins, coated vitamin C, concentrated bacterial suspension and organic selenium pretreatment product are added to water in sequence, then a pH buffer system is added to adjust the pH, and the mixture is sealed to obtain the composite additive.

[0027] The steps described above are described in detail below.

[0028] In step 1, the conditions for the vitamin E microemulsion treatment are as follows: O / W microemulsions were prepared using a nonionic surfactant self-emulsification method. The specific formulation and steps are as follows: the oil phase was vitamin E (α-tocopherol), the surfactant was polyoxyethylene monoalkyl ether (POE-10 lauryl ether), the co-surfactant was anhydrous ethanol, and the aqueous phase was deionized water, which was added slowly dropwise.

[0029] The ratio of vitamin E, polyoxyethylene monoalkyl ether, anhydrous ethanol and water is (5-15):(15-25):(5-15):60.

[0030] Preferably, the ratio of vitamin E, polyoxyethylene monoalkyl ether, anhydrous ethanol, and water is 10:20:10:60. The specific steps of microemulsification are as follows: First, vitamin E (VE) is mixed with a surfactant and a co-surfactant at 50°C to obtain a transparent oily mother liquor; then, an aqueous phase (water) is slowly added dropwise at 35-40°C while stirring at 800 rpm. After a pale blue opalescence appears, stirring continues for 10 minutes. Finally, the mixture is filtered through a 0.22µm membrane to obtain a VE microemulsion with an average particle size of 20-40 nm. After being stored at room temperature for 6 months, it shows no stratification or oxidative acid odor.

[0031] The microemulsification conditions for the 25-hydroxyvitamin D3 are as follows: O / W microemulsions were prepared using a nonionic surfactant self-emulsification method. The specific formulation and steps are as follows: The oil phase consisted of 25-hydroxyvitamin D3 (25-hydroxyvitamin D3 crystals were first dissolved in medium-chain triglycerides (MCT) at 50°C, with the mass of MCT being 5 times that of 25-hydroxyvitamin D3), the surfactant was polyoxyethylene castor oil EL-40 (HLB13), the co-surfactant was 1,2-propanediol, and the aqueous phase was deionized water, which was added slowly dropwise.

[0032] The mass ratio of 25-hydroxyvitamin D3, medium-chain triglycerides (MCT), polyoxyethylene castor oil EL-40 (HLB13), 1,2-propanediol, and deionized water is 1:(4-6):(15-20):(5-10):(65-70).

[0033] Preferably, the mass ratio of 25-hydroxyvitamin D3, medium-chain triglyceride (MCT), polyoxyethylene castor oil EL-40 (HLB13), 1,2-propanediol, and deionized water is 1:5:18:8:68.

[0034] The specific steps of the 25-hydroxyvitamin D3 microemulsion treatment are as follows: 25-hydroxyvitamin D3 was dissolved in medium-chain triglycerides (MCT) at 50°C. Then, surfactants and co-surfactants were added and mixed at 50°C to obtain a transparent oily mother liquor. Aqueous phase (water) was slowly added dropwise at 35°C with stirring at 800 rpm and stirring for 10 min. After filtration, a clear 25-hydroxyvitamin D3 microemulsion with a particle size of about 30 nm was obtained. The content decreased by less than 3% after 3 months of storage at 4°C in the dark.

[0035] The conditions for water-soluble microencapsulation of vitamin A are as follows: A one-step cold-water-dispersible VA microcapsule powder can be obtained using a gelatin-carboxymethyl cellulose (CMC) composite coagulation method. Specific process parameters are as follows: For the microencapsulation wall material: a 1% (g / mL) gelatin solution and a 1% (g / mL) CMC solution are mixed at a mass ratio of (4-6):1, preferably 5:1; then NaCl is added (0.1% of the gelatin solution mass) to enhance the density of the capsule wall. The mass ratio of vitamin A to the wall material is 1:(7-9), preferably 1:8. Sucrose ester, an emulsifier, is also added during the microencapsulation process, with the amount of sucrose ester accounting for 2-5% of the total mass of vitamin A and the wall material.

[0036] Preferably, the amount of sucrose ester added accounts for 3% of the total mass of vitamin A and wall material. The specific preparation process of microencapsulation is as follows: vitamin A and sucrose ester are first dispersed in gelatin solution and sheared at high speed for 10 min; then 10% citric acid solution is added dropwise to adjust the pH of the system to 3.5, CMC-NaCl solution is added under slow stirring, and homogenization is continued for 30 min at a homogenization pressure of 40-60 MPa; then, the mixture is kept at 50℃ for 30 min to complete the composite coagulation, and the pH is adjusted to 6.0 with NaOH; finally, 12% (TG, accounting for the total mass of wall material) transglutaminase is added for cross-linking for 30 min; finally, the mixture is freeze-dried at -70℃ for 24 h, and after pulverization, water-dispersible VA microcapsules are obtained with a particle size of 1-50 μm, an encapsulation rate of ≈81%, and the aqueous solution is milky white suspension without oil precipitation.

[0037] A water-soluble vitamin phase is obtained by mixing VE microemulsion, 25-hydroxyvitamin D3 microemulsion and VA microcapsules, which can be directly compounded with liquid Clostridium butyricum without oil layer precipitation.

[0038] In step 2, the mass ratio of vitamin C to ethyl cellulose is (35-40):(5-10).

[0039] Preferably, the mass ratio of vitamin C to ethyl cellulose is 37:8. The ethyl cellulose is used as a wall material.

[0040] The conditions for spray coating are as follows: the temperature is raised to 80-85°C and refluxed at this temperature for 1-2 hours to completely dissolve the ethyl cellulose wall material in cyclohexane and uniformly disperse vitamin C. Then, the temperature is lowered to 30-40°C by decreasing the temperature by 1-3°C every 5 minutes to allow the ethyl cellulose to separate and precipitate on the surface of vitamin C, forming microcapsules.

[0041] Preferably, the spray coating conditions are as follows: the temperature is raised to 80°C and refluxed at that temperature for 1.5 h, and then the temperature is reduced to 35°C at a rate of 2°C every 5 min, so that ethyl cellulose separates and precipitates on the surface of vitamin C to form microcapsules.

[0042] The vacuum drying conditions are as follows: the residual solvent is removed at 30-40°C to obtain a white coated powder of 40-200 mesh, which yields ethyl cellulose coated vitamin C.

[0043] Preferably, the vacuum drying conditions are as follows: the residual solvent is removed at 35°C to obtain vitamin C coated with 40-200 mesh ethyl cellulose.

[0044] In step 3, the fermentation conditions are as follows: Clostridium butyricum and Bacillus licheniformis are statically cultured at 33-41°C for 24-48 h, the oxygen content is controlled to be less than 0.1 mL / L, the oxygen partial pressure is maintained at <0.1%, and the pH during fermentation is 6-7.

[0045] Preferably, the fermentation conditions are as follows: Clostridium butyricum and Bacillus licheniformis are statically cultured at 37°C for 36 hours, the oxygen content is controlled to be less than 0.1 mL / L, the oxygen partial pressure is maintained at <0.1%, and the pH during fermentation is 6.5.

[0046] Both Clostridium butyricum and Bacillus licheniformis can grow within a temperature range of 33–41°C, but the highest cell concentration is observed at 37°C. The cells reach a stationary phase after 24–48 hours of static incubation; a higher viable cell count or spore yield can be obtained after 36 hours of static incubation.

[0047] This invention requires the use of anaerobic tanks, anaerobic gas generators, liquid paraffin, or other methods to cover or continuously introduce oxygen-free N2 (0.3-0.4 L / min) to maintain an oxygen partial pressure of <0.1%; or to achieve this through biological and chemical oxygen removal methods.

[0048] During fermentation, the pH needs to be maintained at 6-7. A pH of 6.5 is most favorable for cell growth and butyric acid production. A pH below 5.5 will significantly inhibit metabolism and is not conducive to fermentation.

[0049] In step 4, the organic yeast selenium is subjected to ultrasonic disruption combined with enzymatic hydrolysis, which can significantly improve the release rate and bioavailability of selenomethionine.

[0050] The conditions for ultrasonic fragmentation are as follows: ultrasonic energy 300-1500 J, ultrasonic mode is pulsed ultrasound, ultrasound for 3-5 s with an interval of 2-4 s. Pulsed ultrasound can avoid overheating and protect heat-sensitive components. Ultrasound time is 10-20 min. Ultrasound is performed in an ice-water bath to keep the temperature <10℃, which can effectively prevent oxidation.

[0051] Preferably, the conditions for ultrasonic fragmentation are: ultrasonic energy of 1000 J, ultrasonic mode of pulsed ultrasound, ultrasound for 4 seconds, interval of 3 seconds, ultrasound time of 15 minutes, ultrasound in an ice water bath, and maintaining the ultrasonic temperature at 5°C.

[0052] The enzymatic hydrolysis conditions are as follows: enzymatic hydrolysis is performed using a protease at a concentration of 0.1–0.3 mg / mg, the buffer solution is a Tris-HCl buffer solution with a concentration of 0.05–0.2 mol / L, the pH is maintained at 7–8, the water bath shaking frequency is 100–200 r / min, the hydrolysis temperature is 35–40℃, the hydrolysis time is 2–4 h, and the number of hydrolysis cycles is 2–4.

[0053] Preferably, the enzymatic hydrolysis conditions are as follows: enzymatic hydrolysis is performed using a protease at a concentration of 0.2 mg / mg, the buffer solution is a 0.1 mol / L Tris-HCl buffer, the pH is maintained at 7.5, the water bath shaking frequency is 150 r / min, the hydrolysis temperature is 37℃, the hydrolysis time is 3 h, and the hydrolysis is performed 3 times.

[0054] At the above-mentioned concentration of protease, complete enzymatic hydrolysis can be ensured. The added buffer solution maintains the hydrolysis under specific pH conditions, preserving enzyme activity and preventing interference with analysis. The optimal hydrolysis temperature is 37°C, at which point enzyme activity is highest and the hydrolysis effect is best. This invention incorporates water bath agitation during the hydrolysis process, which enhances mass transfer and improves hydrolysis efficiency. Furthermore, multiple hydrolysis cycles increase the release rate and improve the extraction of effective components after hydrolysis.

[0055] In step 5, the pH is adjusted to 6-6.5, preferably to 6.2. The method of using the composite additive of the present invention includes: diluting the composite additive with water at a volume ratio of 1:(1000-2000), using it continuously for 3-5 days, and using it 1-2 times per month.

[0056] The beneficial effects of this invention are as follows: (1) This invention solves the problem of easy oxidation and degradation of vitamins in aqueous systems in traditional additives by using targeted encapsulation and formulation technologies. Specifically, the fat-soluble vitamins A and E in this invention are encapsulated using water-soluble microencapsulation technology, 25-hydroxyvitamin D3 is encapsulated using nanoemulsion technology, and vitamin C is spray-coated using ethyl cellulose as the wall material. Combined with a glycerol-sorbitol composite stabilizer and buffer system, the oxidation half-life of VA, VE, and 25-OH-D3 in aqueous phase is extended from about 30 days in traditional liquid formulations to ≥300 days. After 6 months of accelerated testing at 40°C, the VA retention rate still reached 82%, which is 112% higher than the unencapsulated control group. This invention achieves long-term stable storage of high-dose fat-soluble vitamins in liquid systems for the first time, ensuring the vitamin bioactivity of the liquid product within its 12-month shelf life (25°C), and solving the problem of unstable health care effects caused by vitamin deactivation in traditional powders and ordinary liquid formulations.

[0057] (2) This invention prepares 25-OH-D3 into 80-120 nm nanoemulsions, overcoming the influence of feed oils and effectively improving the absorption rate of 25-OH-D3. It can be directly absorbed in the upper jejunum via the lymphatic and portal vein dual channels. The time to peak blood concentration is shortened from 10-12 h to 3.1 h, and the bioavailability reaches 188%. After continuous feeding of this compound additive for 7 days, the eggshell thickness increases by 0.04 mm or more, and the egg breakage rate decreases by 35% or more, which is significantly better than the existing technology and provides technical support for prolonging the peak egg production period.

[0058] (3) This invention constructs a liquid probiotic protection platform, which, combined with a precise culture process, allows the bacteria to enter a vitrified dormant state (aw=0.89). After storage at 25℃ for 12 months, the survival rate of Clostridium butyricum in this compound additive is ≥85%. After administration via drinking water, the concentration of butyric acid in the ileum increases by 2.3 times, and the detection of toxins decreases by 90%, effectively improving the intestinal microecology of laying hens and solving the problems of rapid decline and low colonization rate of traditional probiotics.

[0059] (4) This invention pretreats yeast selenium through ultrasound-enzymatic hydrolysis, increasing the release of selenomethionine by 42% and simultaneously forming an antioxidant synergistic ring with vitamin E. The GSH-Px activity in laying hen serum reaches 380 U / mL, and the selenium content in egg yolk increases to 0.51 mg / kg (meeting the standard for selenium-enriched eggs). Even after 7 days of discontinuation, the selenium content in egg yolk remains ≥0.3 mg / kg. The pretreatment method described in this invention effectively solves the problem of low organic selenium deposition efficiency, enhances antioxidant capacity, and endows selenium-enriched eggs with higher production value.

[0060] (5) The composite additive described in this invention achieves multi-component co-loading and ternary synergistic effects, simultaneously upgrading the three major systems of laying hens. Experimental verification shows that the egg production rate increases by 5.3 percentage points, the daily egg weight increases by 3.8g / bird, the feed conversion ratio decreases by 0.11, and the mortality rate is significantly reduced, significantly improving the overall breeding benefits and achieving the goal of "high production, superior shell quality, selenium enrichment, and low mortality".

[0061] (6) The composite additive of the present invention is a clear microemulsion with a viscosity of 28 mPa·s. It can be diluted with water at a ratio of 1:1000 to 2000 and can pass through a 0.15 mm water line without clogging, making it suitable for automated aquaculture. The composite additive can be administered via drinking water instead of feed, reducing labor intensity, ensuring uniform feeding, improving aquaculture management efficiency, and meeting the needs of large-scale aquaculture.

[0062] (7) The liquid compound additive of the present invention is non-toxic, with heavy metal content ≤ 1 / 10 of the feed additive limit, selenium residue meets the standard after 0 days of drug withdrawal, and there is no withdrawal period. This compound additive can reduce dust pollution by administering it through drinking water, which meets the requirements of green farming. Detailed Implementation

[0063] The present invention will now be described in detail, and its features and advantages will become clearer and more apparent from these descriptions.

[0064] Example The present invention is further illustrated below with specific examples. These embodiments are merely illustrative and not intended to limit the scope of the invention. All raw materials used in the embodiments of the present invention are commercially available.

[0065] Example 1 The dosage of each ingredient in 1L of compound additive is as follows: Vitamin A 700,000 IU, Vitamin C 7 g, Vitamin E 3 g, 25-hydroxyvitamin D3 3 g, and Butyric acid bacteria (1.5-2) × 10 9 CFU / mL (with viable count ≥1 billion), Bacillus licheniformis (5.5–6) × 10 8 Weigh out the following raw materials: CFU / mL (with viable bacterial count ≥ 500 million), 0.8 g of organic selenium, and 12 g of stabilizer. The amount of pH buffer added should be sufficient to adjust the pH of the compound additive to 6.0–6.5, and the amount of water added should be sufficient to bring the compound additive to 1 L.

[0066] A method for preparing a composite additive, and a liquid composite additive obtained therefrom, the preparation method comprising the following steps: Vitamin A was subjected to water-soluble microencapsulation. The water-soluble microencapsulation method was as follows: Raw material preparation: Microencapsulation wall material: 1% (g / mL) gelatin solution and 1% (g / mL) CMC solution were mixed at a mass ratio of 5:1; then NaCl was added (the amount of NaCl added was 0.1% of the mass of the gelatin solution) to enhance the density of the capsule wall. The mass ratio of vitamin A to wall material was 1:8. Sucrose ester, an emulsifier, was also added during the microencapsulation process, with the amount of sucrose ester accounting for 3% of the total mass of vitamin A and wall material. The specific processing method is as follows: First, disperse the above-mentioned vitamin A and sucrose ester in a gelatin solution and shear at high speed for 10 min; then add 10% citric acid solution to adjust the pH of the system to 3.5, add CMC-NaCl solution under slow stirring, and continue homogenization for 30 min at a homogenization pressure of 40-60 MPa; then keep warm at 50℃ for 30 min to complete the composite coagulation, and then adjust the pH to 6.0 with NaOH; finally, add 12% (TG, accounting for the total mass of the wall material) transglutaminase for cross-linking for 30 min; finally, freeze dry at -70℃ for 24 h, and after pulverization, obtain water-dispersible VA microcapsules with a particle size of 1-50 μm, an encapsulation rate of ≈81%, and an aqueous solution that is milky white and suspended without oil precipitation.

[0067] Vitamin E was microemulsified. The microemulsification method was as follows: Raw material preparation: The ratio of vitamin E, polyoxyethylene monoalkyl ether, anhydrous ethanol, and water was 10:20:10:60. The specific microemulsification steps were as follows: First, vitamin E was mixed with surfactant and co-surfactant at 50°C to obtain a transparent oily mother liquor; then, aqueous phase (water) was slowly added dropwise at 35-40°C while stirring at 800 rpm. After a pale blue opalescence appeared, stirring was continued for 10 minutes. Finally, the mixture was filtered through a 0.22µm membrane to obtain a vitamin E microemulsion with an average particle size of 20-40 nm. After being stored at room temperature for 6 months, it showed no stratification and no oxidative acid odor.

[0068] 25-hydroxyvitamin D3 was microemulsified. The microemulsification method was as follows: Raw material preparation: The mass ratio of 25-hydroxyvitamin D3, medium-chain triglycerides (MCT), polyoxyethylene castor oil EL-40 (HLB13), 1,2-propanediol, and deionized water was 1:5:18:8:68. The specific microemulsification steps were as follows: 25-hydroxyvitamin D3 was dissolved in MCT at 50°C, then a surfactant and co-surfactant were added and mixed at 50°C to obtain a transparent oily mother liquor. Aqueous phase (water) was slowly added dropwise at 35°C with a stirring speed of 800 rpm and stirred for 10 min. After filtration, a clear 25-hydroxyvitamin D3 microemulsion with a particle size of approximately 30 nm was obtained. The content decreased by <3% after 3 months of storage at 4°C in the dark. The vitamin E microemulsion, 25-hydroxyvitamin D3 microemulsion, and vitamin A microcapsules were mixed to obtain a water-soluble vitamin phase.

[0069] Vitamin C and ethyl cellulose were accurately weighed according to a mass ratio of 37:8. The mixture was heated to 80°C and refluxed at this temperature for 1.5 h to completely dissolve the ethyl cellulose in cyclohexane and uniformly disperse the vitamin C. Subsequently, the temperature was lowered to 35°C at a rate of 2°C every 5 min, causing the ethyl cellulose to separate and precipitate on the surface of the vitamin C, forming microcapsules. The residual solvent was then dried at 35°C to obtain vitamin C coated with ethyl cellulose of 40–200 mesh.

[0070] Clostridium butyricum and Bacillus licheniformis were cultured statically at 37°C for 36 h, with the oxygen content controlled to be less than 0.1 mL / L, the oxygen partial pressure maintained at <0.1%, and the pH during fermentation at 6.5. The oxygen content was controlled by continuously introducing anaerobic N2 (0.3–0.4 L / min). After fermentation, the bacterial cells were collected by centrifugation, and a concentrated bacterial suspension was prepared using a glycerol-sorbitol composite stabilizer.

[0071] The organic yeast selenium was ultrasonically disrupted under the following conditions: ultrasonic energy of 1000 J, ultrasonic mode of pulsed ultrasound, ultrasonication for 4 s followed by a 3 s interval, and ultrasonication time of 15 min, in an ice-water bath at a temperature of 5°C. After ultrasonic disruption, enzymatic hydrolysis was performed under the following conditions: a protease at a concentration of 0.2 mg / mg was used, the buffer solution was 0.1 mol / L Tris-HCl, the pH was maintained at 7.5, the water bath shaking frequency was 150 r / min, the hydrolysis temperature was 37°C, the hydrolysis time was 3 h, and the hydrolysis was repeated 3 times.

[0072] Under nitrogen protection, water-soluble vitamins, coated vitamin C, concentrated bacterial suspension and organic selenium pretreatment product are added to water in sequence. Then, a pH buffer system (sodium citrate + phosphate) is added to adjust the pH to 6.2. The mixture is then sealed to obtain a liquid composite additive.

[0073] Acute oral toxicity tests on this compound additive showed that the LD50 (in mice) was... 50 >5000mg / kg indicates that the compound additive is practically non-toxic; the content of heavy metals (Pb, As, Cd, Hg) is ≤1 / 10 of the feed additive limit; after 0 days of withdrawal, the selenium residue in each tissue is lower than the pollution-free standard of NY / T1954-2010, and no withdrawal period is required.

[0074] Example 2 The dosage of each ingredient in 1L of the compound additive is as follows: Vitamin A 500,000 IU, Vitamin C 5 g, Vitamin E 2 g, 25-hydroxyvitamin D3 2.5 g, and Butyric acid bacteria (1.5-2) × 10 9CFU / mL (with viable count ≥1 billion), Bacillus licheniformis (5.5–6) × 10 8 CFU / mL (with viable bacteria count ≥ 500 million), 0.5 g of organic selenium, 10 g of stabilizer, the amount added to the pH buffer system should be sufficient to adjust the pH of the compound additive to 6.0-6.5, and the amount of water added should be sufficient to add the compound additive to 1L.

[0075] A method for preparing a composite additive, and a liquid composite additive obtained therefrom, the preparation method comprising the following steps: Vitamin A was microencapsulated for water solubility. The water-soluble microencapsulation process was the same as in Example 1. Vitamin E was then microemulsified. The microemulsification process was the same as in Example 1. 25-hydroxyvitamin D3 was then microemulsified. The microemulsification process was the same as in Example 1. The vitamin E microemulsion, 25-hydroxyvitamin D3 microemulsion, and vitamin A microcapsules were mixed to obtain the water-soluble vitamin phase.

[0076] Vitamin C and ethyl cellulose were accurately weighed according to a mass ratio of 35:5. The mixture was heated to 80°C and refluxed at this temperature for 2 hours to completely dissolve the ethyl cellulose in cyclohexane and uniformly disperse the vitamin C. Subsequently, the temperature was lowered to 40°C at a rate of 1°C every 5 minutes, causing the ethyl cellulose to separate and precipitate on the surface of the vitamin C, forming microcapsules. The residual solvent was then dried at 40°C to obtain vitamin C coated with ethyl cellulose of 40–200 mesh.

[0077] Clostridium butyricum and Bacillus licheniformis were cultured statically at 33°C for 48 h, with the oxygen content controlled to be less than 0.1 mL / L and the oxygen partial pressure maintained at <0.1%. The pH during fermentation was 6–7, and the oxygen content was controlled by continuously introducing anaerobic N2 (0.3–0.4 L / min). After fermentation, the bacterial cells were collected by centrifugation, and a concentrated bacterial suspension was prepared using a glycerol-sorbitol composite stabilizer.

[0078] The organic yeast selenium was ultrasonically disrupted under the following conditions: ultrasonic energy 300 J, ultrasonic mode pulsed ultrasonication, ultrasonication for 5 s followed by a 2 s interval, and ultrasonication time for 20 min, all performed in an ice-water bath at a temperature <10℃. Following ultrasonic disruption, enzymatic hydrolysis was performed under the following conditions: protease concentration 0.1 mg / mg, buffer solution 0.05 mol / L Tris-HCl, pH maintained at 7–8, water bath shaking frequency 100 r / min, hydrolysis temperature 35℃, hydrolysis time 4 h, and hydrolysis repeated 4 times.

[0079] Under nitrogen protection, water-soluble vitamins, coated vitamin C, concentrated bacterial suspension and organic selenium pretreatment product are added to water in sequence, and then a pH buffer system (sodium citrate + phosphate) is added to adjust the pH to 6. After sealing, the liquid composite additive is obtained. Example 3 The dosage of each ingredient in 1L of compound additive is as follows: Vitamin A 1,000,000 IU, Vitamin C 10 g, Vitamin E 5 g, 25-hydroxyvitamin D3 4 g, and Butyric acid bacteria (1.5-2) × 10 9 CFU / mL (with viable count ≥1 billion), Bacillus licheniformis (5.5–6) × 10 8 CFU / mL (with viable bacteria count ≥ 500 million), 1.0 g of organic selenium, 15 g of stabilizer, the amount of pH buffer added should be sufficient to adjust the pH of the compound additive to 6.0-6.5, and the amount of water added should be sufficient to add the compound additive to 1 L.

[0080] A method for preparing a composite additive, and a liquid composite additive obtained therefrom, the preparation method comprising the following steps: Vitamin A was microencapsulated for water solubility. The water-soluble microencapsulation process was the same as in Example 1. Vitamin E was then microemulsified. The microemulsification process was the same as in Example 1. 25-hydroxyvitamin D3 was then microemulsified. The microemulsification process was the same as in Example 1. The vitamin E microemulsion, 25-hydroxyvitamin D3 microemulsion, and vitamin A microcapsules were mixed to obtain the water-soluble vitamin phase.

[0081] Vitamin C and ethyl cellulose were accurately weighed at a mass ratio of 40:10. The mixture was heated to 85°C and refluxed at this temperature for 1 hour to completely dissolve the ethyl cellulose in cyclohexane and uniformly disperse the vitamin C. Subsequently, the temperature was lowered by 3°C every 5 minutes until it reached 30°C, causing the ethyl cellulose to separate and precipitate on the surface of the vitamin C, forming microcapsules. The residual solvent was then dried at 30°C to obtain vitamin C coated with ethyl cellulose of 40–200 mesh.

[0082] Clostridium butyricum and Bacillus licheniformis were cultured statically at 41°C for 24 h, with the oxygen content controlled to be less than 0.1 mL / L and the oxygen partial pressure maintained at <0.1%. The pH during fermentation was 6–7, and the oxygen content was controlled by continuously introducing anaerobic N2 (0.3–0.4 L / min). After fermentation, the bacterial cells were collected by centrifugation, and a concentrated bacterial suspension was prepared using a glycerol-sorbitol composite stabilizer.

[0083] The organic yeast selenium was ultrasonically disrupted under the following conditions: ultrasonic energy of 1500 J, ultrasonic mode of pulsed ultrasonication, ultrasonication for 3 s followed by a 4 s interval, and ultrasonication time of 10 min, in an ice-water bath at a temperature <10℃. After ultrasonic disruption, enzymatic hydrolysis was performed under the following conditions: protease concentration of 0.3 mg / mg, buffer solution of 0.2 mol / L Tris-HCl, pH maintained at 7–8, water bath shaking frequency of 200 r / min, hydrolysis temperature of 40℃, hydrolysis time of 2 h, and hydrolysis repeated twice.

[0084] Under nitrogen protection, water-soluble vitamins, coated vitamin C, concentrated bacterial suspension and organic selenium pretreatment product are added to water in sequence. Then, a pH buffer system (sodium citrate + phosphate) is added to adjust the pH to 6.5. After sealing, the liquid composite additive is obtained. Comparative Example Comparative Example 1 The liquid composite additive was prepared in a manner similar to that in Example 1, except that vitamin A was not microencapsulated. Comparative Example 2 The liquid composite additive was prepared in a manner similar to that of Example 1, except that vitamin C was not coated. Comparative Example 3 The liquid composite additive was prepared in a manner similar to that of Example 1, except that the organic yeast selenium was not subjected to ultrasonic disruption.

[0085] Experimental Example Experiment Example 1 Effect Test Laying hens were fed with the liquid compound additives from Examples 1-3 (experimental group), Comparative Examples 1-3 (experimental group), and conventional drinking water (control group) respectively, and tests were conducted. The test procedure was as follows: The liquid compound additive and water were mixed at a volume ratio of 1:1500 and fed to the laying hens via drinking water for 4 consecutive days, once a month. Egg production rate, eggshell strength, mortality rate, and serum selenium content were tested, and the results are shown in Table 1. In Table 1, egg production rate, eggshell strength, mortality rate, and serum selenium content are all obtained by comparing with laying hens fed with conventional drinking water. For example, +25% egg production rate means that compared with laying hens fed with conventional drinking water, the egg production rate increased by 25%.

[0086] Table 1

[0087] As can be seen from Table 1, feeding laying hens with the liquid composite additive described in this invention can effectively improve the laying rate, eggshell strength and selenium content of egg white, while also reducing the mortality rate.

[0088] The present invention has been described in detail above with reference to specific embodiments and exemplary examples; however, these descriptions should not be construed as limiting the present invention. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and embodiments of the present invention without departing from the spirit and scope of the invention, and all such modifications and improvements fall within the scope of the present invention. The scope of protection of the present invention is defined by the appended claims.

Claims

1. A compound additive for the health care of egg-laying poultry, characterized in that, The compound additive includes vitamin A, vitamin C, vitamin E, 25-hydroxyvitamin D3, Clostridium butyricum, Bacillus licheniformis, organic selenium, stabilizer, pH buffer system and water; The amounts of each ingredient in the 1 L composite additive are as follows: Vitamin A 500,000–1,000,000 IU, Vitamin C 5–10 g, Vitamin E 2–5 g, 25-hydroxyvitamin D3 2.5–4 g, and butyric acid bacteria ≥1×10 9 CFU / mL (with viable bacterial count ≥ 1 billion), Bacillus licheniformis ≥ 5 × 10⁻⁶ 8 CFU / mL (with viable bacteria count ≥ 500 million), organic selenium 0.5–1.0 g, stabilizer 10–15 g, the amount of pH buffer system added should be sufficient to adjust the pH of the compound additive to 6.0–6.5, the remainder being water.

2. The compound additive for poultry health care according to claim 1, characterized in that, The amounts of each ingredient in the 1 L composite additive are as follows: Vitamin A 700,000 IU, Vitamin C 7 g, Vitamin E 3 g, 25-hydroxyvitamin D3 3 g, and Butyric acid bacteria ≥1×10 9 CFU / mL (with viable bacterial count ≥ 1 billion), Bacillus licheniformis ≥ 5 × 10⁻⁶ 8 CFU / mL (with viable bacteria count ≥ 500 million), 0.8 g of organic selenium, 12 g of stabilizer, the amount of pH buffer added should be sufficient to adjust the pH of the compound additive to 6.0-6.5, the remainder is water.

3. A method for preparing the composite additive according to claim 1 or 2, characterized in that, The preparation method includes the following steps: Step 1: Vitamin A, Vitamin E, and 25-OH-D3 are processed into water-soluble vitamins by microemulsion or microencapsulation, respectively. Step 2: Spray-coat vitamin C with ethyl cellulose and then vacuum dry to obtain ethyl cellulose-coated vitamin C; Step 3: Ferment Clostridium butyricum and Bacillus licheniformis, collect the bacterial cells by centrifugation, and prepare a concentrated bacterial suspension using glycerol-sorbitol composite stabilizer; Step 4: The organic selenium is subjected to ultrasonic crushing and enzymatic hydrolysis in sequence to obtain the organic selenium pretreated product; Step 5: Under nitrogen protection, water-soluble vitamins, coated vitamin C, concentrated bacterial suspension and organic selenium pretreatment product are added to water in sequence, then a pH buffer system is added to adjust the pH, and the mixture is sealed to obtain the composite additive.

4. The preparation method according to claim 3, characterized in that, In step 1, The microemulsion treatment conditions are as follows: first, vitamin E or 25-OH-D3 is mixed with surfactant and co-surfactant to obtain a transparent oily mother liquor. Then, the aqueous phase is slowly added dropwise while stirring. After a light blue opalescence appears, stirring is continued. Finally, the mixture is filtered through a membrane to obtain a microemulsion.

5. The preparation method according to claim 3, characterized in that, In step 1, The microcapsule processing conditions are as follows: Vitamin A and sucrose esters are first dispersed in a gelatin solution, subjected to high-speed shearing, then citric acid solution is added dropwise to adjust the pH of the system, CMC-NaCl solution is added under slow stirring, homogenization is continued, and then the temperature is maintained to complete the composite coagulation, and then the pH is adjusted with NaOH; finally, transglutaminase is added for cross-linking reaction; finally, after freeze drying and pulverization, water-dispersible VA microcapsules are obtained.

6. The preparation method according to claim 3, characterized in that, In step 2, The mass ratio of vitamin C to ethyl cellulose is (35-40):(5-10); The conditions for spray coating are as follows: the temperature is raised to 80-85°C and refluxed at this temperature for 1-2 hours to completely dissolve the ethyl cellulose wall material in cyclohexane and uniformly disperse vitamin C. Then, the temperature is lowered to 30-40°C by decreasing the temperature by 1-3°C every 5 minutes to allow the ethyl cellulose to separate and precipitate on the surface of vitamin C, forming microcapsules.

7. The preparation method according to claim 3, characterized in that, In step 2, The vacuum drying conditions are as follows: the residual solvent is removed at 30-40°C to obtain a white coated powder of 40-200 mesh, which yields ethyl cellulose coated vitamin C.

8. The preparation method according to claim 3, characterized in that, In step 3, The fermentation conditions are as follows: Clostridium butyricum and Bacillus licheniformis are statically cultured at 33–41°C for 24–48 h, with the oxygen content controlled to be less than 0.1 mL / L, the oxygen partial pressure maintained at <0.1%, and the pH during fermentation being 6–7.

9. The preparation method according to claim 3, characterized in that, In step 4, The conditions for ultrasonic fragmentation are as follows: ultrasonic energy 300-1500 J, ultrasonic mode is pulsed ultrasound, ultrasound for 3-5 s with an interval of 2-4 s. Pulsed ultrasound can avoid overheating and protect heat-sensitive components. Ultrasound time is 10-20 min. Ultrasound is performed in an ice-water bath to keep the temperature <10℃, which can effectively prevent oxidation.

10. The preparation method according to claim 3, characterized in that, In step 4, The enzymatic hydrolysis conditions are as follows: enzymatic hydrolysis is performed using a protease at a concentration of 0.1–0.3 mg / mg, the buffer solution is a Tris-HCl buffer solution with a concentration of 0.05–0.2 mol / L, the pH is maintained at 7–8, the water bath shaking frequency is 100–200 r / min, the hydrolysis temperature is 35–40℃, the hydrolysis time is 2–4 h, and the number of hydrolysis cycles is 2–4.

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