A livestock feed containing a composite probiotic-enzyme preparation and a method for preparing the same

Abstract

The present application belongs to the field of feed additives and animal nutrition, and particularly relates to a kind of compound probiotics-enzyme preparation containing livestock and poultry feed and its preparation method; For the current strain and enzyme system compatibility not strong, bacteria and enzyme ratio disorder leads to poor synergistic effect even appears competitive inhibition or functional antagonism problem; The present application combines probiotics and enzyme preparation according to suitable proportion, and realizes segmented site, release in turn by adopting three-layer core-shell microcapsule structure, the enzyme preparation in the core is embedded in starch-trehalose gel matrix, and is coated with hydroxypropyl methyl cellulose phthalate, the middle layer is hydrophobic fat barrier, the outermost layer is compound probiotics, probiotics is attached to the surface of the middle layer by spray freeze drying, forms porous shell, through dynamic bacteria and enzyme synergistic system, probiotics and enzyme preparation exert synergistic effect at correct time and site, avoid mutual inhibition caused by improper ratio.

Description

Technical Field

[0001] This invention belongs to the field of feed additives and animal nutrition, specifically relating to a livestock and poultry feed containing a compound probiotic-enzyme preparation and its preparation method. Background Technology

[0002] In modern livestock and poultry farming, probiotic preparations and enzyme preparations are two important types of feed additives, playing key roles in improving animal gut health and enhancing nutrient digestibility, respectively. Probiotics can regulate the balance of intestinal microbiota, enhance immunity, and promote nutrient absorption; while enzyme preparations can degrade anti-nutritional factors and macromolecules in feed, improving the availability of feed nutrients. To further improve feeding results, in recent years, researchers have begun to explore the combination of multiple enzyme preparations with multiple strains of probiotics in feed, aiming to achieve synergistic effects. However, the existing "bacteria-enzyme combination" model suffers from problems such as weak specificity of formulation and insufficient coordination of effects, specifically manifested in the following aspects:

[0003] First, there is a lack of design for enzyme-microbe combinations tailored to different stages of the digestive tract. Traditional practices often involve simply mixing and adding various enzymes and probiotics, ignoring the differences in their sites of action and optimal conditions within the animal's digestive tract. For example, phytase requires suitable pH conditions in the upper small intestine to release phosphorus from phytic acid, but the proliferation of many probiotics in the upper intestine may lower the pH or produce organic acids, thus affecting the effectiveness of phytase. Similarly, some cellulases require a longer time to act on the fibrous matrix, while large intestine probiotics mainly colonize the hindgut; if the two are not in the same location, a synergistic effect is unlikely.

[0004] Secondly, an imbalance in the ratio of probiotics to enzyme preparations can easily lead to competitive inhibition or even functional antagonism. Different strains and enzyme systems have different requirements for substrates and environments. If the ratio is inappropriate, the following may occur: an excess of a certain enzyme may destroy the substrate or microenvironment on which probiotics depend for survival; conversely, a large amount of probiotic metabolites may inactivate the enzyme or reduce its efficiency. In addition, enzyme preparations themselves are usually proteins, and some probiotics may produce proteases or other metabolites that degrade the enzyme preparation, causing the functions of both to cancel each other out. These factors can prevent the compound probiotic-enzyme preparation from achieving the expected synergistic effect, and in severe cases, even lead to antagonistic effects that cancel each other out.

[0005] Secondly, existing methods of adding probiotics and enzymes suffer from activity loss during processing and feeding. Feed pelleting is typically carried out at high temperatures, during which enzymes are easily inactivated, and a large number of probiotics die. Even if probiotics are sprayed on after pelleting, the strong action of stomach acid and bile during feeding significantly reduces probiotic survival and enzyme activity. Furthermore, simply mixing and adding enzymes and probiotics may interact during feed storage, affecting their stability.

[0006] In summary, there is an urgent need for a new technical solution that can precisely combine probiotic strains with corresponding enzyme systems for different parts of the digestive tract of livestock and poultry, and release them in a coordinated manner in space and time to achieve the best synergistic effect. At the same time, this solution should ensure the stability of probiotics and enzyme preparations during feed processing and passage through the gastrointestinal tract, avoiding functional loss caused by improper ratios or premature contact, thereby effectively solving the problems of weak targeted synergistic effects, imbalanced ratios, and loss of activity in existing technologies. Summary of the Invention

[0007] This invention provides a livestock and poultry feed containing a compound probiotic-enzyme preparation and its preparation method. It aims to overcome the shortcomings of the prior art and achieve precise release and high synergy of probiotics and enzyme preparations in various stages of the digestive tract of livestock and poultry through innovative bacterial-enzyme synergistic design and preparation process, thereby significantly improving nutrient utilization and livestock and poultry production performance.

[0008] The specific technical solution is as follows:

[0009] A livestock and poultry feed containing a compound probiotic-enzyme preparation and its preparation method are as follows:

[0010] S1: Preparation of the kernel enzyme layer.

[0011] S11: Add soluble starch and trehalose to deionized water, stir, cool, and prepare a gel matrix solution.

[0012] S12: Phytase, xylanase, β-glucanase, and acidic protease are mixed and added to the gel matrix solution prepared in S11. Shearing and emulsification are performed simultaneously with the addition to prepare an enzyme-gel mixed suspension.

[0013] S13: Hydroxypropyl methylcellulose phthalate is dissolved in acetone to prepare a coating solution with a concentration of 5-8%; the enzyme-gel mixed suspension prepared in S12 is spray-dried to prepare soluble starch-trehalose gel-embedded primary enzyme microcapsules, and then the primary enzyme microcapsules are coated with the coating solution and dried to obtain core enzyme layer microcapsules.

[0014] S2: Intermediate isolation layer covering.

[0015] S21: Heat the hydrogenated palm oil in a water bath to 60-65°C and stir until it is completely melted into a clear and transparent liquid.

[0016] S22: The kernel enzyme layer microcapsules prepared in S13 are preheated, and the hydrogenated palm oil liquid prepared in S21 is used to coat the preheated kernel enzyme layer microcapsules through fluidized bed coating. After coating, the microcapsules are cooled to room temperature to obtain the ribozyme layer microcapsules with the isolation layer.

[0017] S3: The outer shell of the fungal layer is fixed.

[0018] S31: Under sterile conditions, add gelatin powder to deionized water, stir at 5°C to allow it to swell, then raise the temperature to 55°C and keep stirring until the gelatin is completely dissolved. Add trehalose and stir until completely dissolved. Cool to 4°C to obtain a cryoprotectant solution.

[0019] S32: Under aseptic conditions, Lactobacillus plantarum, Clostridium butyricum, and Bacillus licheniformis are mixed to obtain a mixed bacterial powder; the mixed bacterial powder is stirred and mixed with the cryoprotectant solution prepared in S31 in an ice-water bath at 4°C to obtain a compound probiotic suspension.

[0020] S33: Cool the ribozyme layer microcapsules coated with the isolation layer prepared in S22 to 4°C, and then spray the compound probiotic suspension prepared in S32 onto the surface of the ribozyme layer microcapsules coated with the isolation layer at a low temperature of 4°C. After spraying, immediately perform ultra-low temperature freezing, and after freezing, put them into a vacuum freeze-drying chamber for drying. Discharge the material under the protection of dry nitrogen to obtain core-shell microcapsules.

[0021] S4: Prepare livestock and poultry feed.

[0022] S41: Mix the core-shell microcapsules prepared in S33 with the carrier bran to obtain a primary premix containing bacterial enzyme preparation.

[0023] S42: Mix the primary premix containing probiotic enzyme preparation prepared in S41 with the core feed matrix, then spray in soybean oil, add vitamins, and finally mix, granulate, cool, and package to obtain livestock and poultry feed containing compound probiotic-enzyme preparation.

[0024] Furthermore, in the soluble starch and trehalose described in S11, the soluble starch accounts for 60-80% of the solid mass in the gel matrix, and the trehalose accounts for 20-40% of the solid mass in the gel matrix; the deionized water is such that the total mass of the soluble starch and trehalose accounts for 20-30% of the total mass of the gel matrix solution.

[0025] The stirring parameters described in S11 are: rotation speed 200-300 rpm, duration 20-30 min, and temperature 80-85℃.

[0026] The phytase in S12 has a mass percentage of 45-60%; the xylanase has a mass percentage of 20-30%; the β-glucanase has a mass percentage of 10-20%; the acidic protease has a mass percentage of 5-15%; and the enzyme in the enzyme-gel mixed suspension has a mass percentage of 15-30%.

[0027] The shear emulsification described in S12 has the following parameter settings: rotation speed 5000-8000 rpm, duration 3-5 min.

[0028] The hydroxypropyl methylcellulose phthalate described in S13 accounts for 10-20% of the core mass.

[0029] The spray drying described in S13 has the following parameter settings: inlet air temperature 100-110℃, outlet air temperature 60-70℃, and pressure 0.35-0.45MPa.

[0030] The coating described in S13 has the following parameter settings: preheating temperature 30-35℃, spray gun speed 10-20mL / min.

[0031] The drying process described in S13 has the following parameters: temperature 35-40℃, duration 10-15min.

[0032] Furthermore, the hydrogenated palm oil described in S21 has a melting point of 58–60°C and a mass of 8–12% of the weight of the core microcapsules.

[0033] The preheating described in S22 has a temperature of 35-40°C.

[0034] The fluidized coating described in S22 has the following parameter settings: inlet air temperature 45-50℃, atomization pressure 0.2-0.4MPa, and spray rate 5-15mL / min.

[0035] Furthermore, the low-temperature stirring described in S31 has the following parameter settings: rotation speed 100-200 rpm.

[0036] The cryoprotectant solution described in S31 contains trehalose to deionized water at a mass-to-volume ratio of 8-12%, and gelatin to deionized water at a mass-to-volume ratio of 3-5%.

[0037] The mixed bacterial powder described in S32 has a mass ratio of 1:5 to 1:10 with the cryoprotectant, wherein Lactobacillus plantarum accounts for 40-60% of the mixed bacterial powder, Clostridium butyricum accounts for 25-40% of the mixed bacterial powder, and Bacillus licheniformis accounts for 10-25% of the mixed bacterial powder.

[0038] The ultra-low temperature freezing described in S33 has the following parameter settings: temperature -40 to -35℃, duration 2 to 4 hours.

[0039] The drying process described in S33 has the following parameter settings: the first stage involves heating from -40℃ to -20℃ for 10 hours; the second stage involves heating from -20℃ to 25℃ for 15 hours.

[0040] The core-shell microcapsules described in S33 contain a compound probiotic suspension comprising 10-25% by mass.

[0041] Furthermore, the carrier bran described in S41 has a mass that is 5 to 10 times the mass of the core-shell microcapsule.

[0042] The livestock and poultry feed containing compound probiotic-enzyme preparations as described in S42, wherein the mass percentage of core-shell microcapsules is 0.5-3%, and the mass percentage of feed matrix is ​​97-99.5%.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] 1. This invention achieves a "enzyme first, bacteria later" release sequence through a core-shell structure and pH-sensitive materials, ensuring that the enzymes function before the probiotics, thus creating a favorable environment for probiotic colonization.

[0045] 2. This invention achieves a synergistic effect by precisely matching different segments of the digestive tract, avoiding competitive inhibition.

[0046] 3. This invention maintains the high activity of bacterial enzymes during feed processing and storage by using high-temperature resistant gel encapsulation and cryoprotectants. Attached Figure Description

[0047] Figure 1 This is a livestock and poultry feed containing compound probiotic-enzyme preparations and its preparation process flow diagram.

[0048] Figure 2 This is a comparison chart of the gastric environment enzyme activity retention rate, intestinal environment enzyme activity release rate, and probiotic survival rate data of Examples 1-4 and Comparative Examples 1-2. Detailed Implementation

[0049] The following embodiments further explain and illustrate the technical solutions of the present invention. It should be specifically noted that each specific embodiment is a concretization and explanation of the technical solution and should not be considered as a limitation on the scope of protection of the present invention. Those skilled in the art still have the right to modify the technical solutions of these embodiments and make equivalent substitutions for some or all of the technical features, and these modifications or substitutions do not change the essence of the corresponding technical solutions, nor do they cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions described in the present invention.

[0050] This invention proposes a livestock and poultry feed containing a compound probiotic-enzyme preparation and its preparation method. It employs a three-layer "enzyme-shell" core-shell structure: the core is a compound enzyme preparation containing phytase, xylanase, β-glucanase, acidic protease, etc., uniformly embedded in a heat-resistant soluble starch-trehalose gel matrix, and coated with hydroxypropyl methylcellulose phthalate. The middle layer is a dense, hydrophobic fatty substance, hydrogenated palm oil; the outer layer is a compound probiotic layer containing multiple synergistic probiotics, composed of *Lactobacillus plantarum*, *Clostridium butyricum*, and *Bacillus licheniformis*. The probiotics, after being treated with a cryoprotectant, are attached to the surface of the middle insulating layer using a spray freeze-drying process, forming a porous outer shell layer. (See attached...) Figure 1 The diagram shows a preparation process for livestock and poultry feed containing a compound probiotic-enzyme preparation. The detailed technical solution is as follows:

[0051] 1. Preparation of kernel enzyme layer

[0052] The core objective of the core enzyme layer is to construct a microcapsule system that can effectively protect enzyme activity under the high temperatures and acidic conditions of feed pelleting and rapidly release enzymes into the duodenum. On one hand, trehalose and soluble starch form a glassy gel matrix. At high temperatures, this matrix forms a robust amorphous structure, immobilizing and isolating enzyme molecules to prevent thermal denaturation and inactivation. On the other hand, hydroxypropyl methylcellulose phthalate (HP-55) is used. This material is insoluble in acidic environments; it only begins to dissolve when the pH value is above 5.0, as the phthalate groups in the molecules ionize, thus achieving targeted enzyme release into the duodenum.

[0053] The rationale for using hydroxypropyl methylcellulose phthalate in this invention lies in the fact that its molecular chains require contact with an aqueous solution with a pH > 5.0. However, the core is sealed in an extremely dry environment during storage after preparation and before passing through the stomach. Without sufficient water molecules as a medium, the dissolution reaction of HP-55 cannot be initiated. Simultaneously, since the core is a solid matrix composed of a soluble starch-trehalose gel, and the enzyme preparation is encapsulated in this inert gel matrix in solid form, both are very stable. Therefore, a dry, non-reactive environment is maintained within the membrane until the triggering conditions are met.

[0054] Choosing an inlet air temperature of 100–110°C for spray drying will not lead to a significant loss of enzyme activity. After the enzyme-gel mixture is atomized into countless micron-sized droplets, its surface area increases dramatically. Upon entering the high-temperature airflow, the water on the surface of the droplets evaporates instantly. This evaporation process absorbs a large amount of heat, making the temperature inside the droplets much lower than the temperature of the hot air. At the same time, the gel formed by soluble starch and trehalose will quickly form a glassy solid matrix around the enzyme molecules during the drying process, fixing and isolating the enzyme, further shielding it from the direct impact of high temperature.

[0055] 2. Encapsulation of the intermediate isolation layer

[0056] The intermediate insulating layer functions as a physical barrier that can be emulsified by bile. In the stomach: the solid fat layer is stable in the acidic environment of the stomach, and its hydrophobic properties effectively block the penetration of water molecules and hydrogen ions, ensuring that even if a small amount of probiotics on the outer shell are released prematurely, they cannot penetrate this barrier to contact the enzymes in the core, fundamentally avoiding ineffective interactions in the acidic environment of the stomach. In the intestine: when chyme enters the duodenum, bile salts emulsify this fat barrier like a "cleanser." The emulsification process breaks it down from a continuous membrane into tiny fat droplets, thus forming "channels" on the membrane. This process takes 15–30 minutes, providing a valuable time difference for the enzymes in the core to be released and exert their effects under pH triggering. At the same time, by precisely controlling the thickness and melting point of the insulating layer, its emulsification and degradation rate in the intestine can be regulated.

[0057] The reason for using an air intake of 45–50°C is that when molten hydrogenated palm oil is atomized from the spray gun, it enters the fluidized bed chamber as extremely fine droplets. During its very short journey to the material surface, the droplets exchange heat with the cooler airflow, causing their temperature to drop. When these cooled oil droplets collide with the material, whose surface temperature is maintained at 35–40°C, intense heat exchange occurs. Because the material surface temperature is lower than the freezing point of the oil, the droplets solidify instantly or within a very short time upon contact with the material surface, thus coating the surface.

[0058] 3. Fixation of the outer shell fungal layer

[0059] The compound probiotics are firmly fixed to the outer surface of the intermediate insulating layer, forming the shell of the microcapsules. A protective solution composed of trehalose and gelatin, during ultra-low temperature rapid freezing, does not form large ice crystals but undergoes a "glass transition," forming an amorphous, glass-like solid. This solid structure perfectly "fixes" the bacteria in their instantaneous life state, avoiding damage from ice crystals and solutes, maximizing the integrity of the cell structure. Simultaneously, because the enzymes are protected by the HP-55 enteric coating and hydrogenated palm oil as the intermediate insulating layer, ultra-low temperature rapid freezing causes minimal damage to the enzymes. By "spraying" the bacterial suspension onto the surface of the microcapsules coated with the insulating layer and immediately deep-freezing, the bacteria and protective agent are evenly distributed on the surface of each microcapsule in the form of tiny droplets. The subsequent vacuum freeze-drying process removes the ice crystals, leaving a porous, sponge-like network structure that firmly embeds the dormant bacteria within and attaches them to the intermediate layer. This achieves "in-situ immobilization" of the bacteria, rather than simple physical mixing. Furthermore, from the preparation of bacterial suspension and spraying to freeze drying, the entire process is always carried out at low temperatures, completely avoiding heat damage to the bacteria.

[0060] 4. Feed preparation

[0061] The prepared core-shell microcapsules are physically mixed with the core feed matrix to achieve uniform distribution of functional additives in the feed.

[0062] To avoid the difficulty of uniform mixing when micro-additives are directly added to large quantities of feed, a gradient dilution method is adopted, starting with small amounts and gradually increasing the concentration. First, a high concentration of microcapsules is premixed with a small amount of carrier to form a "premix," which is then gradually expanded to include more feed ingredients, ensuring statistically uniform distribution. Although the core-shell microcapsule structure is reinforced, it is still necessary to avoid damage to its physical structure from high-intensity shear forces. Dry microcapsule powder is prone to static electricity, causing it to adhere to the inner walls of equipment or clump together. By using a small amount of liquid carrier, such as soybean oil, static electricity can be neutralized, flowability improved, and the adsorption effect of the carrier particles can be utilized to ensure uniform adhesion of the microcapsules to their surface, achieving more stable dispersion.

[0063] Example 1

[0064] A method for preparing livestock and poultry feed containing a compound probiotic-enzyme preparation is as follows:

[0065] Table 1 Main Raw Materials

[0066]

[0067]

[0068] S1: Preparation of the kernel enzyme layer.

[0069] S11: Add soluble starch and trehalose to deionized water, stir, and cool to prepare a gel matrix solution. The total mass of soluble starch and trehalose accounts for 25% of the total mass of the gel matrix solution, with soluble starch accounting for 70% and trehalose accounting for 30%. Stirring parameters are set as follows: speed 250 rpm, duration 25 min, temperature 83℃.

[0070] S12: Phytase, xylanase, β-glucanase, and acidic protease are mixed and added to the gel matrix solution prepared in S11. Shearing and emulsification are performed simultaneously with the addition to prepare an enzyme-gel mixed suspension. The mass percentages of phytase, xylanase, β-glucanase, and acidic protease in the enzyme-gel mixed suspension are 53%, 25%, 15%, and 7%, respectively. The enzyme powder accounts for 23% of the total mass of the enzyme-gel mixed suspension.

[0071] S13: Hydroxypropyl methylcellulose phthalate was dissolved in acetone to prepare a 6.5% coating solution. The enzyme-gel mixture suspension prepared in S12 was spray-dried to prepare soluble starch-trehalose gel-encapsulated primary enzyme microcapsules. The primary enzyme microcapsules were then coated with the coating solution and dried to obtain core enzyme layer microcapsules. Hydroxypropyl methylcellulose phthalate accounted for 15% of the core mass. The spray drying parameters were: inlet air temperature 105℃, outlet air temperature 65℃, and pressure 0.40MPa. The coating parameters were: preheating temperature 33℃ and spray gun speed 15mL / min.

[0072] S2: Intermediate isolation layer covering.

[0073] S21: Heat the hydrogenated palm oil in a water bath to 63°C and stir until completely melted into a clear, transparent liquid. The mass of the hydrogenated palm oil is 10% of the weight of the core microcapsules.

[0074] S22: The ribozyme layer microcapsules prepared in S13 were preheated to 38℃, and the hydrogenated palm oil liquid prepared in S21 was used to coat the preheated ribozyme layer microcapsules through fluidized bed coating. After coating, the microcapsules were cooled to room temperature to obtain ribozyme layer microcapsules with an isolation layer. The fluidized bed coating parameters were set as follows: inlet air temperature 48℃, atomization pressure 0.3MPa, and spray rate 10mL / min.

[0075] S3: The outer shell of the fungal layer is fixed.

[0076] S31: Under aseptic conditions, gelatin powder is added to deionized water and stirred at 5°C and 150 rpm until swollen. The temperature is then raised to 55°C and stirred until the gelatin is completely dissolved. Trehalose is then added and stirred until completely dissolved. The solution is cooled to 4°C to obtain a cryoprotectant solution. The mass-to-volume ratio of trehalose to deionized water is 10%, and the mass-to-volume ratio of gelatin to deionized water is 4%.

[0077] S32: Under aseptic conditions, *Lactobacillus plantarum*, *Clostridium butyricum*, and *Bacillus licheniformis* are mixed to obtain a mixed bacterial powder. The mixed bacterial powder is then stirred and mixed with the cryoprotectant solution prepared in S31 at 4°C in an ice-water bath to obtain a compound probiotic suspension. The mixture contains 50% *Lactobacillus plantarum*, 33% *Clostridium butyricum*, and 17% *Bacillus licheniformis*. The mass ratio of the mixed bacterial powder to the cryoprotectant is 1:8.

[0078] S33: The ribozyme layer microcapsules coated with the isolation layer prepared in S22 are cooled to 4℃. Then, the composite probiotic suspension prepared in S32 is sprayed onto the surface of the ribozyme layer microcapsules coated with the isolation layer at a low temperature of 4℃. After spraying, they are immediately subjected to ultra-low temperature freezing. After freezing, they are placed in a vacuum freeze-drying chamber for drying. The material is discharged under the protection of drying nitrogen to obtain core-shell microcapsules. The ultra-low temperature freezing parameters are set as follows: temperature -38℃, duration 3h, and the composite probiotic suspension accounts for 15% of the mass of the core-shell microcapsules.

[0079] S4: Prepare livestock and poultry feed.

[0080] S41: The core-shell microcapsules prepared in S33 are mixed with the carrier bran to obtain a primary premix containing the bacterial enzyme preparation. The bran has a mass 8 times that of the core-shell microcapsules.

[0081] S42: The primary premix containing the microbial enzyme preparation prepared in S41 is mixed with the core feed matrix, then soybean oil is sprayed in, followed by vitamins. Finally, the mixture is mixed, granulated, cooled, and packaged to obtain livestock and poultry feed containing the compound probiotic-enzyme preparation. The core-shell microcapsules account for 1.5% of the total mass, and the core feed matrix accounts for 98.5% of the total mass.

[0082] Example 2

[0083] The composition and preparation process are the same as in Example 1, except that:

[0084] The soluble starch and trehalose in S11 of the preparation process are wherein the soluble starch accounts for 60% of the solid mass of the gel matrix and the trehalose accounts for 40% of the solid mass of the gel matrix; the deionized water is such that the total mass of the soluble starch and trehalose accounts for 20% of the total mass of the gel matrix solution, and the other components are the same.

[0085] In step S11 of the preparation process, the stirring parameters are set as follows: rotation speed 200 rpm, duration 20 min, temperature 80℃, and other steps are the same.

[0086] In the preparation process S12, phytase accounts for 45% by mass; xylanase accounts for 30% by mass; β-glucanase accounts for 20% by mass; acidic protease accounts for 5% by mass; and enzyme powder accounts for 15% by mass in the enzyme-gel mixed suspension. Other components are the same.

[0087] In step S12 of the preparation process, shear emulsification is performed with the following parameters: rotation speed 5000 rpm, duration 3 min, and other steps are the same.

[0088] In the preparation process, hydroxypropyl methylcellulose phthalate in S13 accounts for 10% of the core mass. Hydroxypropyl methylcellulose phthalate is dissolved in acetone to prepare a 5% coating solution, with other components remaining the same.

[0089] In step S13 of the preparation process, the spray drying parameters are set as follows: inlet air temperature 100℃, outlet air temperature 60℃, and pressure 0.35MPa; the coating parameters are set as follows: preheating temperature 30℃ and spray gun speed 10mL / min; the drying parameters are set as follows: temperature 35℃ and duration 10min, with other steps being the same.

[0090] In step S21 of the preparation process, the hydrogenated palm oil is heated to 60°C in a water bath, and the other steps are the same.

[0091] In the preparation process S21, the mass of hydrogenated palm oil is 8% of the weight of the core microcapsules, and the other components are the same.

[0092] In step S22 of the preparation process, the preheating temperature is 35℃, and the fluidized coating parameters are set as follows: inlet air temperature 45℃, atomization pressure 0.2MPa, and spray rate 5mL / min; other steps are the same.

[0093] In step S31 of the preparation process, the stirring speed is 100 rpm at low temperature, and the other steps are the same.

[0094] In the S31 preparation process, the cryoprotectant solution contains 8% trehalose and 3% gelatin and deionized water by mass volume, with other components being the same.

[0095] In the preparation process S32, Lactobacillus plantarum accounts for 40%, Clostridium butyricum accounts for 40%, Bacillus licheniformis accounts for 20%, the mass ratio of mixed bacterial powder to cryoprotectant is 1:5, the mass ratio of compound probiotic suspension in core-shell microcapsules is 10%, and other components are the same.

[0096] The ultra-low temperature freezing parameters in S33 of the preparation process are set as follows: temperature -40℃, duration 2h, and other steps are the same.

[0097] In the preparation process S41, the carrier bran has a mass that is 5 times that of the core-shell microcapsules, and the other components are the same.

[0098] The livestock and poultry feed containing compound probiotic-enzyme preparation in preparation process S42 has the following composition: the mass ratio of core-shell microcapsules is 0.5%, the mass ratio of core feed matrix is ​​99.5%, and the other components are the same.

[0099] Example 3

[0100] The composition and preparation process are the same as in Example 1, except that:

[0101] The soluble starch and trehalose in S11 of the preparation process are wherein the soluble starch accounts for 80% of the solid mass of the gel matrix and the trehalose accounts for 20% of the solid mass of the gel matrix; the deionized water is such that the total mass of the soluble starch and trehalose accounts for 30% of the total mass of the gel matrix solution, and the other components are the same.

[0102] In step S11 of the preparation process, the stirring parameters are set as follows: rotation speed 300 rpm, duration 30 min, temperature 85℃, and other steps are the same.

[0103] In the preparation process S12, phytase accounts for 60% by mass; xylanase accounts for 20% by mass; β-glucanase accounts for 12% by mass; acidic protease accounts for 8% by mass; and enzyme powder accounts for 30% by mass in the enzyme-gel mixed suspension. Other components are the same.

[0104] In step S12 of the preparation process, shear emulsification is performed with the following parameters: rotation speed 8000 rpm, duration 5 min, and other steps are the same.

[0105] In the preparation process, hydroxypropyl methylcellulose phthalate in S13 accounts for 20% of the core mass. Hydroxypropyl methylcellulose phthalate is dissolved in acetone to prepare a coating solution with a concentration of 8%, and other components are the same.

[0106] In the preparation process S13, the spray drying parameters are set as follows: inlet air temperature 110℃, outlet air temperature 70℃, and pressure 0.45MPa; the coating parameters are set as follows: preheating temperature 35℃ and spray gun speed 20mL / min; the drying parameters are set as follows: temperature 40℃ and duration 15min, with other steps being the same.

[0107] In step S21 of the preparation process, the hydrogenated palm oil is heated to 65°C in a water bath, and the other steps are the same.

[0108] In process S21, the mass of hydrogenated palm oil is 12% of the weight of the core microcapsules, and the other components are the same.

[0109] In step S22 of the preparation process, the preheating temperature is 40℃, and the fluidized coating parameters are set as follows: inlet air temperature 50℃, atomization pressure 0.4MPa, and spray rate 15mL / min; other steps are the same.

[0110] In step S31 of the preparation process, the stirring speed is 200 rpm at low temperature, and the other steps are the same.

[0111] In the S31 preparation process, the cryoprotectant solution contains trehalose at a mass-to-volume ratio of 12% to deionized water, gelatin at a mass-to-volume ratio of 5% to deionized water, and other components are the same.

[0112] In the preparation process S32, Lactobacillus plantarum accounts for 60%, Clostridium butyricum accounts for 25%, Bacillus licheniformis accounts for 15%, the mass ratio of mixed curing agent to cryoprotectant is 1:10, the mass ratio of compound probiotic suspension in core-shell microcapsules is 25%, and other components are the same.

[0113] The ultra-low temperature freezing parameters in S33 of the preparation process are set as follows: temperature -35℃, duration 4h, and other steps are the same.

[0114] In the preparation process S41, the carrier bran has a mass that is 10 times that of the core-shell microcapsules, and the other components are the same.

[0115] The livestock and poultry feed containing compound probiotic-enzyme preparation in preparation process S42 has a mass ratio of 3% for core-shell microcapsules and 97% for core feed matrix, with other components being the same.

[0116] Example 4

[0117] The composition and preparation process are the same as in Example 1, except that:

[0118] The soluble starch and trehalose in S11 of the preparation process are wherein the soluble starch accounts for 75% of the solid mass of the gel matrix and the trehalose accounts for 25% of the solid mass of the gel matrix; the deionized water is such that the total mass of the soluble starch and trehalose accounts for 22% of the total mass of the gel matrix solution, and the other components are the same.

[0119] In step S11 of the preparation process, the stirring parameters are set as follows: rotation speed 280 rpm, duration 24 min, temperature 84℃, and other steps are the same.

[0120] In the preparation process S12, phytase accounts for 55% by mass; xylanase accounts for 22% by mass; β-glucanase accounts for 17% by mass; acidic protease accounts for 6% by mass; and enzyme powder accounts for 20% by mass in the enzyme-gel mixed suspension. Other components are the same.

[0121] In step S12 of the preparation process, shear emulsification is performed with the following parameters: rotation speed 7000 rpm, duration 3.5 min, and other steps are the same.

[0122] In the preparation process, hydroxypropyl methylcellulose phthalate in S13 accounts for 16% of the core mass. Hydroxypropyl methylcellulose phthalate is dissolved in acetone to prepare a 7% coating solution, with other components remaining the same.

[0123] In step S13 of the preparation process, the spray drying parameters are set as follows: inlet air temperature 108℃, outlet air temperature 66℃, and pressure 0.38MPa; the coating parameters are set as follows: preheating temperature 34℃ and spray gun speed 12mL / min; the drying parameters are set as follows: temperature 39℃ and duration 14min, with other steps being the same.

[0124] In step S21 of the preparation process, the hydrogenated palm oil is heated to 64°C in a water bath, and the other steps are the same.

[0125] In the preparation process S21, the mass of hydrogenated palm oil is 9% of the weight of the core microcapsules, and the other components are the same.

[0126] In step S22 of the preparation process, the preheating temperature is 36℃, and the fluidized coating parameters are set as follows: inlet air temperature 46℃, atomization pressure 0.25MPa, and spray rate 12mL / min; other steps are the same.

[0127] In step S31 of the preparation process, the stirring speed is 120 rpm at low temperature, and the other steps are the same.

[0128] In the S31 preparation process, the cryoprotectant solution contains 9% trehalose and 3.5% gelatin and deionized water by mass volume, with other components being the same.

[0129] In the preparation process S32, Lactobacillus plantarum accounts for 43%, Clostridium butyricum accounts for 32%, Bacillus licheniformis accounts for 25%, the mass ratio of mixed bacterial powder to cryoprotectant is 1:9, the mass ratio of compound probiotic suspension in core-shell microcapsules is 20%, and other components are the same.

[0130] The ultra-low temperature freezing parameters in S33 of the preparation process are set as follows: temperature -39℃, duration 2.5 seconds, and other steps are the same.

[0131] In the preparation process S41, the carrier bran has a mass that is 6 times that of the core-shell microcapsules, and the other components are the same.

[0132] The livestock and poultry feed containing compound probiotic-enzyme preparation in preparation process S42 has a mass ratio of 1.2% for core-shell microcapsules and 98.8% for core feed matrix, with other components being the same.

[0133] Comparative Example 1

[0134] The composition and preparation process are the same as in Example 1, except that:

[0135] The compound probiotics are encapsulated in microcapsules, and the enzyme preparation is encapsulated in the outer layer, with the other steps being the same.

[0136] Comparative Example 2

[0137] The enzyme preparation and probiotics were mixed and added to the feed according to the traditional method, and the amount of enzyme preparation and probiotics added was the same as in Example 1.

[0138] Based on Examples 1-4 and Comparative Examples 1-2, samples of microcapsule samples or directly mixed samples were taken for gastric environment enzyme activity retention tests: Microcapsules were placed in a buffer solution containing pepsin at pH 2.0–3.0 and incubated at 37°C with constant shaking for 2 hours. After removal, the pH was adjusted to the optimal range for the enzyme to terminate the pepsin reaction. After centrifugation, the supernatant was collected, and the remaining enzyme activity was measured. The retention rate was calculated by comparing the enzyme activity with that of the untreated sample. After the gastric environment digestion was completed, the sample was transferred to a buffer solution containing trypsin at pH 6.8–7.2 and incubated for another 3 hours. After centrifugation, the supernatant was collected, and the released enzyme activity was measured. The release rate was calculated by comparing the enzyme activity with that of the sample after the gastric environment digestion was completed, referring to the standard GB / T 44967-2024 "General Rules for Enzyme Preparations for Feed".

[0139] Based on Examples 1-4 and Comparative Examples 1-2, samples were taken from the microcapsules before and after gastric juice treatment to test the survival rate of probiotics. The plate count method was used. The samples were appropriately diluted with sterile physiological saline, spread, and cultured under anaerobic conditions for 48 hours. The survival rate was obtained by counting the number of colonies formed and calculating the ratio of the number of viable bacteria after treatment to the number of viable bacteria at the beginning.

[0140] Based on Examples 1-4 and Comparative Examples 1-2, the prepared feed samples were sampled and tested for feed dry matter digestibility. The feed samples were pulverized and passed through a 40-mesh sieve. They were first digested with chicken pepsin at 37°C and pH 2, and then digested with chicken trypsin at 37°C and pH 6.8. After digestion, the residue was filtered, centrifuged at 4000 rpm for 20 min, and the undigested residue was collected, dried to constant weight, and the feed dry matter digestibility was calculated.

[0141] Based on Examples 1-4 and Comparative Examples 1-2, samples of the prepared feed were taken for average daily weight gain testing. Seven treatment groups were established, each containing 12 chicks (half male and half female) of a commercially available AA white-feathered broiler breed, aged 1 day old, of the same breed, in good health, and with no significant difference in initial weight. A standard two-stage broiler diet was used. The experiment was conducted in a chicken house, strictly following the broiler rearing manual, controlling temperature, humidity, lighting programs, and ventilation to reduce environmental stress. A 3-day pre-trial period was established to allow the chicks to acclimatize to the chicken house environment, followed by a 42-day formal trial period. Fasting weight was measured on the first day of the formal trial period and again on the last day of the formal trial period to obtain the average daily weight gain.

[0142] The specific test results are shown in Table 2:

[0143] Table 2 Comparison of core performance of Examples 1-4 and Comparative Examples 1-2

[0144]

[0145] The above comparison results show that Example 1 exhibits the best overall performance, largely due to the three-layer structure of the microcapsule: the core enzyme layer is effectively protected in the acidic environment of the stomach; the intermediate isolation layer ensures that the enzyme and bacteria do not interfere with each other during storage and in the stomach; and the outer bacterial layer targets and releases probiotics in the intestine. This programmed release mechanism fundamentally avoids competition or inhibition between bacteria and enzymes at inappropriate times and locations. This demonstrates that Example 1 successfully addresses the imbalance in the bacterial-enzyme ratio caused by insufficient specificity in strain and enzyme compatibility. Probiotics and enzyme preparations need to be in an appropriate ratio to achieve the best synergistic effect; an improper ratio may lead to... The two exhibit competitive inhibition or even functional antagonism. The overall performance of Examples 2 to 4 is slightly lower than that of Example 1, but still remains at a high level, indicating that excellent synergistic effects of bacteria and enzymes were still achieved under a wide range of parameter variations. In contrast, the enzyme and bacteria in Comparative Example 1 lacked synergy, and the feed dry matter digestibility and daily weight gain were significantly reduced. The enzyme in Comparative Example 2 was rapidly released in the intestine, but because it was largely inactivated in the stomach, the total effective enzyme activity was low. Without the protection of microcapsules, gastric acid and proteases would severely destroy the activity of the bacterial enzymes. At the same time, the bacterial enzymes were prematurely contacted in the stomach and could not reach the intestine to exert their effects.

Claims

1. A livestock and poultry feed containing a compound probiotic-enzyme preparation, characterized in that: The livestock and poultry feed comprises conventional feed base components and compound probiotic-enzyme preparation microcapsules; the compound probiotic-enzyme preparation microcapsules have a core-shell structure, comprising, from the inside out: a core enzyme layer, an intermediate isolation layer, and an outer bacterial layer; the core enzyme layer is composed of a compound enzyme preparation embedded in a soluble starch-trehalose gel matrix and coated with a layer of hydroxypropyl methylcellulose phthalate enteric material, the intermediate isolation layer is a lipid material, and the outer bacterial layer is a compound probiotic layer; The livestock and poultry feed is prepared as follows: S1: Preparation of the kernel enzyme layer; S11: Add soluble starch and trehalose to deionized water, stir, cool, and prepare a gel matrix solution; S12: Phytase, xylanase, β-glucanase, and acidic protease are mixed and added to the gel matrix solution prepared in S11. Shearing and emulsification are performed while adding the mixture to prepare an enzyme-gel mixed suspension. S13: Hydroxypropyl methylcellulose phthalate is dissolved in acetone to prepare a coating solution with a concentration of 5-8%; the enzyme-gel mixed suspension prepared in S12 is spray-dried to prepare soluble starch-trehalose gel-embedded primary enzyme microcapsules, and then the primary enzyme microcapsules are coated with the coating solution and dried to obtain core enzyme layer microcapsules. S2: Intermediate isolation layer covering; S21: Heat the hydrogenated palm oil in a water bath to 60-65°C and stir until it is completely melted into a clear and transparent liquid. S22: The kernel enzyme layer microcapsules prepared in S13 are preheated, and the hydrogenated palm oil liquid prepared in S21 is used to coat the preheated kernel enzyme layer microcapsules through fluidized coating. After coating, the microcapsules are cooled to room temperature to obtain the ribozyme layer microcapsules with the isolation layer. S3: The outer shell bacterial layer is fixed; S31: Under sterile conditions, gelatin powder is added to deionized water and stirred at 5°C to make it swell. Then the temperature is raised to 55°C and stirred until the gelatin is completely dissolved. Trehalose is added and stirred until completely dissolved. The solution is cooled to 4°C to obtain a cryoprotectant solution. S32: Under aseptic conditions, Lactobacillus plantarum, Clostridium butyricum, and Bacillus licheniformis are mixed to obtain a mixed bacterial powder; the mixed bacterial powder is stirred and mixed with the cryoprotectant solution prepared in S31 in an ice-water bath at 4°C to obtain a compound probiotic suspension. S33: Cool the ribozyme layer microcapsules coated with the isolation layer prepared in S22 to 4°C, and then spray the compound probiotic suspension prepared in S32 onto the surface of the ribozyme layer microcapsules coated with the isolation layer at a low temperature of 4°C. After spraying, immediately perform ultra-low temperature freezing, and after freezing, put them into a vacuum freeze-drying chamber for drying. Discharge the material under the protection of dry nitrogen to obtain core-shell microcapsules. S4: Preparation of livestock and poultry feed; S41: Mix the core-shell microcapsules prepared in S33 with the carrier bran to obtain a primary premix containing bacterial enzyme preparation; S42: Mix the primary premix containing probiotic enzyme preparation prepared in S41 with the core feed matrix, then spray in soybean oil, add vitamins, and finally mix, granulate, cool, and package to obtain livestock and poultry feed containing compound probiotic-enzyme preparation.

2. The livestock and poultry feed containing a compound probiotic-enzyme preparation according to claim 1, characterized in that: The compound enzyme preparation comprises phytase, xylanase, β-glucanase, and acidic protease, with the following mass percentages: phytase 45-60%; xylanase 20-30%; β-glucanase 10-20%; and acidic protease 5-15%.

3. The livestock and poultry feed containing a compound probiotic-enzyme preparation according to claim 1, characterized in that: The lipid material is hydrogenated palm oil with a melting point of 58–60°C.

4. The livestock and poultry feed containing a compound probiotic-enzyme preparation according to claim 1, characterized in that: The composite probiotic layer, which accounts for 10-25% of the mass of the core-shell microcapsule, includes Lactobacillus plantarum, Clostridium butyricum and Bacillus licheniformis, with the following mass percentages: Lactobacillus plantarum 40-60%, Clostridium butyricum 25-40%, and Bacillus licheniformis 10-25%.

5. The livestock and poultry feed containing a compound probiotic-enzyme preparation according to claim 1, characterized in that: The soluble starch and trehalose described in S11, wherein the soluble starch accounts for 60-80% of the solid mass of the gel matrix, and the trehalose accounts for 20-40% of the solid mass of the gel matrix; the deionized water is such that the total mass of the soluble starch and trehalose accounts for 20-30% of the total mass of the gel matrix solution; The stirring described in S11 has the following parameters: rotation speed 200-300 rpm, duration 20-30 min, and temperature 80-85℃. The enzyme mass percentage in the enzyme-gel mixed suspension described in S12 is 15-30%; The shear emulsification described in S12 has the following parameter settings: rotation speed 5000-8000 rpm, duration 3-5 min.

6. The livestock and poultry feed containing a compound probiotic-enzyme preparation according to claim 1, characterized in that: The hydroxypropyl methylcellulose phthalate described in S13 accounts for 10-20% of the mass of the core. The spray drying described in S13 has the following parameter settings: inlet air temperature 100-110℃, outlet air temperature 60-70℃, and pressure 0.35-0.45MPa. The coating described in S13 has the following parameter settings: preheating temperature 30-35℃, spray gun speed 10-20mL / min; The drying process described in S13 has the following parameters: temperature 35-40℃, duration 10-15min.

7. The livestock and poultry feed containing a compound probiotic-enzyme preparation according to claim 1, characterized in that: The hydrogenated palm oil described in S21 has a mass of 8-12% of the mass of the core microcapsules; The preheating described in S22 has a temperature of 35-40°C; The fluidized coating described in S22 has the following parameter settings: inlet air temperature 45-50℃, atomization pressure 0.2-0.4MPa, and spray rate 5-15mL / min.

8. The livestock and poultry feed containing a compound probiotic-enzyme preparation according to claim 1, characterized in that: The low-temperature stirring described in S31 has the following parameter settings: rotation speed 100-200 rpm; In the cryoprotectant solution described in S31, the mass-volume ratio of trehalose to deionized water is 8-12%, and the mass-volume ratio of gelatin to deionized water is 3-5%. The mixed bacterial powder described in S32 has a mass ratio of 1:5 to 1:10 with the cryoprotectant. The ultra-low temperature freezing described in S33 has the following parameter settings: temperature -40 to -35°C, duration 2 to 4 hours; The drying process described in S33 has the following parameter settings: the first stage involves heating from -40℃ to -20℃ for 10 hours; the second stage involves heating from -20℃ to 25℃ for 15 hours.

9. The livestock and poultry feed containing a compound probiotic-enzyme preparation according to claim 1, characterized in that: The carrier bran described in S41 has a mass that is 5 to 10 times the mass of the core-shell microcapsule; The livestock and poultry feed containing compound probiotic-enzyme preparations as described in S42, wherein the mass percentage of core-shell microcapsules is 0.5-3%, and the mass percentage of feed matrix is ​​97-99.5%.

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