A composite probiotic type livestock growth promoting feed additive and a preparation method thereof

Abstract

This invention relates to the field of feed additive technology, and discloses a compound probiotic-type growth-promoting feed additive for livestock and poultry and its preparation method. The additive is made from calcium-based bentonite, sodium hexametaphosphate, D-trehalose dihydrate, L-ascorbic acid sodium, carboxymethyl cellulose sodium powder, gluconate-δ-lactone powder, and *Lactobacillus plantarum* fermentation concentrate. During the room-temperature preparation stage, sodium hexametaphosphate complexes calcium ions, blocking their early cross-linking with carboxymethyl cellulose sodium, maintaining the fluidity of the slurry to ensure uniform dispersion of the bacteria. During the drying and heating stage, gluconate-δ-lactone hydrolyzes to produce acid, lowering the pH of the system, and the released calcium ions form a cross-linking network with carboxymethyl cellulose sodium in situ. This product has compressive strength, can withstand the mechanical stress during granulation, and slows down steam heat transfer, improving the survival rate of *Lactobacillus plantarum* in granulation processing and room-temperature, high-humidity storage environments.

Description

Technical Field

[0001] This invention relates to the field of feed additive technology, specifically to a compound probiotic-type growth-promoting feed additive for livestock and poultry and its preparation method. Background Technology

[0002] Currently, probiotics such as Lactobacillus plantarum are often formulated into live bacteria feed additives due to their ability to regulate the gut microbiota and promote growth in livestock and poultry. However, these additives face the problem of rapid decline in live bacteria count due to high processing intensity and variable storage conditions in practical applications. Therefore, it is necessary to encapsulate and protect the probiotics with specific polysaccharides, proteins, or inorganic mineral matrices to reduce the negative impact of environmental factors on bacterial activity.

[0003] Regarding the aforementioned issues, existing probiotic preservation technologies typically employ materials such as gelatin and dextrin as wall materials. The probiotic fermentation broth is directly mixed with these macromolecules and then dehydrated via high-temperature spray drying. Alternatively, conventional acidic substances or metal salt curing agents are added to the matrix formulation. Free ions in the solution directly contact polymers such as sodium carboxymethyl cellulose during a room-temperature physical mixing stage, causing cross-linking reactions in the macromolecules during stirring. This is followed by conventional mechanical stirring, drying, and pulverization.

[0004] The existing process has several shortcomings. First, the cross-linking process of the materials during the pulping stage cannot be controlled; at room temperature, free ions directly contact the polymer and undergo early reactions, leading to a sharp increase in slurry viscosity and localized clumping, making it difficult to achieve uniform dispersion of the bacterial solution. Second, the resulting product has low mechanical compressive strength and is prone to breakage under the high stress conditions of feed ring die extrusion. Furthermore, the material has poor heat conduction mitigation effects, and high-temperature steam can easily cause thermal inactivation of the core microorganisms. In addition, the environmental barrier effect of the protective layer is limited, making it prone to moisture absorption and rehydration during room temperature and high humidity storage. The macromolecular chains, lacking rigid locking, loosen and deform, allowing moisture and oxygen to permeate through the internal pores, accelerating the decay rate of the probiotics.

[0005] Therefore, the present invention provides a compound probiotic type livestock and poultry growth-promoting feed additive and its preparation method to overcome the shortcomings of the prior art. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a compound probiotic-type livestock and poultry growth-promoting feed additive and its preparation method, which solves the problems of low survival rate of live bacteria due to insufficient pressure resistance and heat resistance during extrusion pelleting of existing probiotic feed additives, as well as the decline in the number of live bacteria due to easy moisture absorption and oxidation during storage.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a compound probiotic-type growth-promoting feed additive for livestock and poultry, employing the following technical solution:

[0009] A compound probiotic-based growth-promoting feed additive for livestock and poultry is made from raw materials comprising the following parts by weight:

[0010] 15-25 parts of calcium-based bentonite;

[0011] Sodium hexametaphosphate 0.5–2.0 parts;

[0012] 20-35 parts of D-trehalose dihydrate;

[0013] 2-5 parts of L-ascorbic acid sodium;

[0014] 3-8 parts of sodium carboxymethyl cellulose powder;

[0015] 1-4 parts of glucono-δ-lactone powder;

[0016] 30-50 parts of Lactobacillus plantarum fermentation concentrate;

[0017] The viable cell count concentration of the Lactobacillus plantarum fermentation concentrate is 1×10⁻⁶. 10 ~5×10 10 CFU / mL.

[0018] By employing the above technical solution, this invention utilizes the physical and chemical reaction processes between the components to obtain a composite encapsulation system with high compressive strength and high environmental stability. The specific reaction mechanism is as follows:

[0019] Step 1: In the early mixing and dispersion stage, sodium hexametaphosphate is added to the system as an ion complexing agent to pre-complex and lock the calcium ions released during the hydration of calcium-based bentonite, preventing free calcium ions from undergoing early cross-linking and coagulation with sodium carboxymethyl cellulose, maintaining the macroscopic homogeneous rheological state of the liquid system, and ensuring the uniform distribution of Lactobacillus plantarum.

[0020] Step 2: After entering the drying and heating stage, triggered by thermodynamic temperature, gluconic acid-δ-lactone undergoes a ring-opening hydrolysis reaction to generate gluconic acid, causing in-situ acidification inside the material system.

[0021] Step 3: As the system environment changes to weakly acidic, the complexation equilibrium between sodium hexametaphosphate and calcium ions dissociates, slowly releasing calcium ions. The released calcium ions undergo ion coordination reactions with the sodium carboxymethyl cellulose molecular chain segments distributed in the system, constructing a homogeneous three-dimensional cross-linked network in situ within the material.

[0022] The aforementioned three-dimensional cross-linked network, along with the amorphous glass phase formed by the dehydration of D-trehalose dihydrate and bentonite particles, interpenetrate to form a composite physical matrix. This structure can absorb and dissipate the mechanical compression and shear stress generated during the pelleting process in the feed industry, preventing the coating layer from brittlely disintegrating. Simultaneously, it inhibits the relaxation deformation of polymer chain segments during room temperature storage, blocks the penetration of external water molecules and free oxygen into the core region, and improves the survival rate and retention stability of *Lactobacillus plantarum*.

[0023] Preferably, the method for preparing the Lactobacillus plantarum fermentation concentrate includes:

[0024] Lactobacillus plantarum strains were inoculated into modified MRS liquid medium and subjected to isothermal anaerobic fermentation until the end of the logarithmic growth phase to obtain the fermentation broth.

[0025] The fermentation broth was pumped into a centrifuge for centrifugation, the supernatant was discarded, and the bottom bacterial sludge was collected.

[0026] The bottom layer of bacterial sludge was resuspended using sterile physiological saline to obtain a concentrated fermentation solution of Lactobacillus plantarum.

[0027] By adopting the above technical solutions, the fermentation and enrichment process of probiotic strains was standardized, and a bacterial mud substrate with high viable bacterial count and consistent physiological state was obtained.

[0028] Preferably, when preparing the Lactobacillus plantarum fermentation concentrate, the fermentation tank temperature is set to 35-37°C, the initial pH value is controlled to 6.0-6.5, the constant temperature anaerobic fermentation time is 16-24 hours, the centrifugation speed is 4000-6000 rpm, and the centrifugation time is 10-15 minutes.

[0029] By adopting the above technical solution, the logarithmic growth phase metabolic characteristics of Lactobacillus plantarum are matched, the viable cell density of the fermentation broth is increased, and solid-liquid separation is completed through appropriate centrifugation force, maintaining the structural integrity of the cell membrane.

[0030] Secondly, the present invention provides a method for preparing a compound probiotic-type growth-promoting feed additive for livestock and poultry, using the following technical solution:

[0031] A method for preparing a compound probiotic-based growth-promoting feed additive for livestock and poultry includes the following steps:

[0032] Inject deionized water into the reactor and control the water temperature inside the reactor; start the high shear disperser and add calcium-based bentonite at a uniform speed; maintain the above shear rate, add sodium hexametaphosphate into the system, continue high shear dispersion, turn off the high shear disperser, turn on the conventional paddle agitator, and form a homogeneous suspension.

[0033] Strictly control the temperature of the material system inside the reactor. Under low-speed stirring, add the D-trehalose dihydrate and L-ascorbic acid sodium salt to the reactor in sequence and continue stirring. Slowly and evenly sprinkle in sodium carboxymethyl cellulose powder and gluconate-δ-lactone powder while maintaining stirring. Pump in Lactobacillus plantarum fermentation concentrate and continue mixing and stirring to form a macroscopically homogeneous mixed slurry.

[0034] The resulting mixed slurry is transferred to a vacuum paddle dryer. The temperature of the jacket heating medium is controlled, the vacuum pump is turned on, and the absolute pressure of the system is adjusted to maintain this state and slowly evaporate the moisture. When the solid content of the material reaches the predetermined range, the heating system is adjusted to raise the system temperature, and the vacuum valve is adjusted simultaneously to lower the absolute pressure of the system. This temperature and pressure condition is maintained constant. The system temperature is further increased, and the absolute pressure is further reduced. This state is maintained until the residual moisture content of the material drops to the predetermined range. Then the system vacuum is released to obtain the dried crude product.

[0035] The dried crude product is discharged from the equipment and cooled by cold air circulation or natural cooling. It is then fed into a pulverizer for crushing and sieved through a mechanical screening device. The undersized powder is collected to obtain the compound probiotic type livestock and poultry growth-promoting feed additive.

[0036] By adopting the above technical solution, the physicochemical processes such as material hydration and dispersion, thermally triggered acid production, and in-situ cross-linking and dehydration are integrated, realizing the control of rheological state and the separation of phase change solidification operation, thus ensuring the active retention of probiotics in the processing environment.

[0037] Preferably, the amount of deionized water injected is 100-150 parts by weight, and the water temperature inside the reactor is controlled at 20-30°C; the shear rate is adjusted to 3000-5000 s. -1 The running time after adding the calcium-based bentonite is 20-40 minutes; the addition time window for the sodium hexametaphosphate is 5-10 minutes, and the high-shear dispersion time is 15-30 minutes.

[0038] By adopting the above technical solution and setting specific shear rates and water temperatures, it is ensured that the layered silicate mineral particles are fully exfoliated and hydrated, while sodium hexametaphosphate is rapidly introduced into the solution phase to complete the ion complexation reaction, thus preventing the system from agglomerating in the early stage of dispersion.

[0039] Preferably, the temperature of the material system inside the reactor is strictly controlled at 20-25℃; the speed of the conventional paddle agitator is adjusted to 50-100 rpm; the continuous stirring time after adding the D-trehalose dihydrate and L-ascorbic acid sodium is 15-20 minutes; the stirring time after sprinkling the sodium carboxymethyl cellulose powder and glucono-δ-lactone powder is 30-50 minutes; and the continued mixing and stirring time after pumping in the Lactobacillus plantarum fermentation concentrate is 20-40 minutes.

[0040] By adopting the above technical solution, the system temperature is limited to a relatively low temperature range, maintaining the chemical inertness of gluconate-δ-lactone, avoiding early hydrolysis and cross-linking during the macromolecular swelling stage, and ensuring uniform diffusion of Lactobacillus plantarum in a homogeneous low-viscosity fluid.

[0041] Preferably, after the mixed slurry is transferred to the vacuum paddle dryer, the temperature of the jacket heating medium is controlled at 30-38°C, and the absolute pressure of the system is maintained at 8-12 kPa; the time when the solid content of the material reaches the predetermined range refers to when the solid content of the material reaches 55%-65%.

[0042] By adopting the above technical solution, free water is slowly removed in a low-temperature vacuum environment, thereby increasing the solid content of the system and providing the necessary mesoscopic concentration environment for subsequent polymer spatial network reconstruction.

[0043] Preferably, when the solid content of the material reaches 55%–65%, the system temperature is increased to 45–52°C at a heating rate of 1–2°C / min, and the absolute pressure of the system is reduced to 1.5–2.5 kPa. This temperature and pressure condition is maintained constant for 40–90 minutes. The system temperature is then further increased to 53–60°C, and the absolute pressure is further reduced to 0.1–0.6 kPa. The phrase "until the residual moisture content of the material drops to a predetermined range" means until the residual moisture content of the material drops to 4%–6%.

[0044] By adopting the above technical solution, the thermodynamic ring-opening hydrolysis mechanism of gluconate-δ-lactone is precisely activated by using programmed temperature rise to match specific material solid content nodes, so that the in-situ acid production process and the removal of bound water occur simultaneously, avoiding the inactivation damage to bacterial cells caused by excessive local thermal stress.

[0045] Preferably, the material is cooled down to 15-25°C.

[0046] By adopting the above technical solution, polymer chain segments under high temperature are shrunk and frozen at low temperature, locking the already formed physical cross-linked network conformation.

[0047] Preferably, the sieving process using the mechanical screening equipment involves passing the material through a 60-100 mesh sieve.

[0048] By adopting the above technical solution, the geometric particle size distribution of the final powder material is limited, ensuring its dispersion uniformity in the downstream feed mixing process.

[0049] This invention provides a compound probiotic-based growth-promoting feed additive for livestock and poultry, and its preparation method. It has the following beneficial effects:

[0050] 1. This invention achieves regulation of the cross-linking process of materials by using a compound of sodium hexametaphosphate and glucono-δ-lactone. During the room-temperature mixing stage, sodium hexametaphosphate complexes the calcium ions released from calcium-based bentonite, blocking the early cross-linking of calcium ions with sodium carboxymethyl cellulose, maintaining the fluidity of the slurry, and facilitating the uniform dispersion of *Lactobacillus plantarum* in the matrix. During the drying and heating stage, the hydrolysis of glucono-δ-lactone produces acid, leading to a decrease in the pH of the system. The released calcium ions react with sodium carboxymethyl cellulose, constructing a cross-linking network in situ within the material, thus avoiding localized clumping caused by premature cross-linking.

[0051] 2. The product of this invention has compressive strength, improving the survival rate of *Lactobacillus plantarum* during feed extrusion pelleting. The composite matrix composed of dehydrated trehalose, bentonite particles, and cross-linked sodium carboxymethyl cellulose can withstand the mechanical compressive stress during the pelleting process, reducing the likelihood of coating layer rupture. This structure also slows down the heat transfer rate from external high-temperature steam to the material's interior, protecting the activity of *Lactobacillus plantarum* during processing.

[0052] 3. The product of this invention improves the storage stability of probiotics under normal temperature and high humidity conditions. The cross-linked network inside the matrix enhances structural rigidity, inhibits the deformation of polymer chains in humid environments, and slows down the hygroscopic softening of the amorphous glassy phase of trehalose. The dense physical structure reduces the porosity of the powder, reduces the penetration of environmental moisture and oxygen into the interior, slows down the oxidation and hydrolytic inactivation rate of the bacteria during storage, and extends the shelf life. Attached Figure Description

[0053] Figure 1 This is a rheological test curve showing the change of shear stress with shear rate for each group of slurries in this invention.

[0054] Figure 2 This is a biaxial dynamic curve showing the drying time versus the core temperature and pH of the system according to the present invention.

[0055] Figure 3 The stress-strain compression test curves of each group of samples in this invention are shown.

[0056] Figure 4 This is a bar chart showing the Log decay values ​​of viable bacteria count for each group of feed additives of the present invention under extreme industrial pelleting conditions.

[0057] Figure 5 This is a line graph showing the change in viable bacteria retention rate over time under accelerated storage conditions at room temperature according to the present invention. Detailed Implementation

[0058] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0059] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0060] Calcium-based bentonite, whose main component is the hydrous layered silicate mineral montmorillonite, has the CAS number 1302-78-9. Its cation exchange capacity is 100 mmol / 100g, and its interlayer spacing is d. 001 It is 1.35nm.

[0061] Sodium hexametaphosphate is a glassy, ​​straight-chain polyphosphate with the molecular formula (NaPO3). n The CAS number is 10124-56-8, and its average degree of polymerization n is 15.

[0062] Sodium carboxymethyl cellulose is a sodium salt of a carboxymethyl etherified derivative of cellulose, with the molecular formula (C6H7O2(OH)2OCH2COONa). n The CAS number is 9004-32-4, its degree of anionic substitution is 0.9, and the dynamic viscosity of its 2% aqueous solution at 25℃ is 500 mPa·s.

[0063] Lactobacillus plantarum, purchased from the China General Microbiological Culture Collection Center, is a commercially available standard freeze-dried microbial strain.

[0064] The CAS number for D-trehalose dihydrate is 6138-23-4, the CAS number for L-ascorbic acid sodium is 134-03-2, the CAS number for gluconate-δ-lactone is 90-80-2, and the modified MRS liquid culture medium and sterile physiological saline are all commercially available reagents.

[0065] Preparation Examples 1-3:

[0066] Preparation Example 1:

[0067] This preparation example provides a method for preparing Lactobacillus plantarum fermentation concentrate, including the following steps:

[0068] Lactobacillus plantarum strains were inoculated into modified MRS liquid medium and subjected to isothermal anaerobic fermentation. The fermentation tank temperature was set at 35℃, the initial pH was controlled at 6.0, and the isothermal anaerobic fermentation time was 16 hours. The fermentation broth was obtained by culturing to the end of the logarithmic growth phase.

[0069] The fermentation broth was pumped into a centrifuge for centrifugation at a speed of 4000 rpm for 10 minutes. The supernatant was discarded and the bottom bacterial sludge was collected.

[0070] The bottom bacterial sludge was resuspended using sterile physiological saline, and the viable cell concentration of the resuspended solution was adjusted to 1×10⁻⁶. 10 The concentration of CFU / mL was used to prepare a concentrated fermentation solution of Lactobacillus plantarum. The concentrated solution was immediately transferred to a 2°C environment for refrigeration and storage.

[0071] Preparation Example 2:

[0072] This preparation example provides a method for preparing Lactobacillus plantarum fermentation concentrate, including the following steps:

[0073] Lactobacillus plantarum was inoculated into a modified MRS liquid medium and subjected to isothermal anaerobic fermentation. The fermentation tank temperature was set at 36°C, the initial pH was controlled at 6.2, and the isothermal anaerobic fermentation time was 20 hours. The fermentation broth was obtained by culturing to the end of the logarithmic growth phase.

[0074] The fermentation broth was pumped into a centrifuge for centrifugation at a speed of 5000 rpm for 12 minutes. The supernatant was discarded and the bottom bacterial sludge was collected.

[0075] The bottom bacterial sludge was resuspended using sterile physiological saline, and the viable cell count of the resuspended solution was adjusted to 3 × 10⁻⁶. 10 The concentration of CFU / mL was used to prepare a concentrated fermentation solution of Lactobacillus plantarum. The concentrated solution was immediately transferred to a 5°C environment for refrigeration and storage.

[0076] Preparation Example 3:

[0077] This preparation example provides a method for preparing Lactobacillus plantarum fermentation concentrate, including the following steps:

[0078] Lactobacillus plantarum strains were inoculated into modified MRS liquid medium and subjected to isothermal anaerobic fermentation. The fermentation tank temperature was set at 37°C, the initial pH was controlled at 6.5, and the isothermal anaerobic fermentation time was 24 hours. The fermentation broth was obtained by culturing to the end of the logarithmic growth phase.

[0079] The fermentation broth was pumped into a centrifuge for centrifugation at a speed of 6000 rpm for 15 minutes. The supernatant was discarded and the bottom bacterial sludge was collected.

[0080] The bottom bacterial sludge was resuspended using sterile physiological saline, and the viable cell count of the resuspended solution was adjusted to 5 × 10⁻⁶. 10 The concentration of CFU / mL was used to prepare a concentrated fermentation solution of Lactobacillus plantarum. The concentrated solution was immediately transferred to an 8°C environment for refrigeration and storage.

[0081] Examples 1-3:

[0082] Example 1:

[0083] This embodiment provides a compound probiotic-type growth-promoting feed additive for livestock and poultry and its preparation method, including the following steps:

[0084] Inject 100 parts by weight of deionized water into the reactor, turn on the reactor jacket cooling system, and control the water temperature inside the reactor at 20°C; start the high-shear disperser and adjust the shear rate to 3000 s. -1 15 parts by weight of the calcium-based bentonite were added at a uniform rate and run for 20 minutes to hydrate the bentonite particles. While maintaining the above shear rate, 0.5 parts by weight of the sodium hexametaphosphate were slowly added to the system within a 5-minute time window. High shear dispersion was continued for 15 minutes. After the viscosity of the system decreased due to thixotropy, the high shear device was turned off and the conventional paddle agitator was turned on. The speed was adjusted to 50 rpm to form a homogeneous suspension.

[0085] Adjust the flow rate of the coolant in the reactor jacket and strictly control the temperature of the material system inside the reactor at 20°C. Under low-speed stirring at 50 rpm, add 20 parts by weight of the D-trehalose dihydrate and 2 parts by weight of L-ascorbic acid sodium in sequence to the reactor and continue stirring for 15 minutes to dissolve them. Slowly and evenly sprinkle in 3 parts by weight of the sodium carboxymethyl cellulose powder and 1 part by weight of gluconate-δ-lactone powder and maintain stirring for 30 minutes to ensure that the polymer materials are completely swollen. Pump in 30 parts by weight of the Lactobacillus plantarum fermentation concentrate obtained in Preparation Example 1, and continue mixing and stirring for 20 minutes to form a macroscopically homogeneous mixed slurry.

[0086] The resulting mixed slurry was transferred to a vacuum paddle dryer. The temperature of the jacket heating medium was controlled at 30°C. The vacuum pump was turned on, and the absolute pressure of the system was adjusted to maintain at 8 kPa. The moisture was slowly evaporated under this condition. When the solid content of the material reached the predetermined range (i.e., when the solid content of the material reached 55%), the heating system was adjusted to raise the system temperature to 45°C at a heating rate of 1°C / min. At the same time, the vacuum valve was adjusted to reduce the absolute pressure of the system to 1.5 kPa. This temperature and pressure condition was maintained for 40 minutes. The system temperature was then raised to 53°C, and the absolute pressure was further reduced to 0.1 kPa. This condition was maintained until the residual moisture content of the material dropped to the predetermined range (i.e., to 6%). Then the vacuum of the system was released to obtain the dried crude product.

[0087] The dried crude product is discharged from the equipment and cooled down to 15°C by cold air circulation or natural cooling. It is then fed into a pulverizer for crushing and sieved through a mechanical screening device (through a 60-mesh sieve). The powder under the sieve is collected to obtain the compound probiotic type livestock and poultry growth-promoting feed additive.

[0088] Example 2:

[0089] This embodiment provides a compound probiotic-type growth-promoting feed additive for livestock and poultry and its preparation method, including the following steps:

[0090] Inject 125 parts by weight of deionized water into the reactor, turn on the reactor jacket cooling system, and control the water temperature inside the reactor at 25°C; start the high-shear disperser and adjust the shear rate to 4000 s. -1 20 parts by weight of the calcium-based bentonite were added at a uniform rate and run for 30 minutes to hydrate the bentonite particles. While maintaining the above shear rate, 1.2 parts by weight of the sodium hexametaphosphate were slowly added to the system within a 7.5-minute time window. High shear dispersion was continued for 22.5 minutes. After the viscosity of the system decreased due to thixotropy, the high shear device was turned off and a conventional paddle agitator was turned on. The speed was adjusted to 75 rpm to form a homogeneous suspension.

[0091] Adjust the flow rate of the coolant in the reactor jacket and strictly control the temperature of the material system inside the reactor at 22.5℃. Under low-speed stirring at 75 rpm, add 28 parts by weight of the D-trehalose dihydrate and 3.5 parts by weight of L-ascorbic acid sodium in sequence to the reactor and continue stirring for 17.5 minutes to dissolve them. Slowly and evenly sprinkle in 5.5 parts by weight of the sodium carboxymethyl cellulose powder and 2.5 parts by weight of gluconate-δ-lactone powder and maintain stirring for 40 minutes to ensure that the polymer materials are completely swollen. Pump in 40 parts by weight of the Lactobacillus plantarum fermentation concentrate obtained in Preparation Example 2, and continue mixing and stirring for 30 minutes to form a macroscopically homogeneous mixed slurry.

[0092] The resulting mixed slurry was transferred to a vacuum paddle dryer. The temperature of the jacket heating medium was controlled at 34°C. The vacuum pump was turned on, and the absolute pressure of the system was adjusted to maintain 10 kPa. The moisture was slowly evaporated under this condition. When the solid content of the material reached the predetermined range (i.e., when the solid content of the material reached 60%), the heating system was adjusted to raise the system temperature to 48.5°C at a heating rate of 1.5°C / min. At the same time, the vacuum valve was adjusted to reduce the absolute pressure of the system to 2.0 kPa. This temperature and pressure condition was maintained for 65 minutes. The system temperature was then raised to 56.5°C, and the absolute pressure was further reduced to 0.35 kPa. This condition was maintained until the residual moisture content of the material dropped to the predetermined range (i.e., dropped to 5%). Then the vacuum of the system was released to obtain the dried crude product.

[0093] The dried crude product is discharged from the equipment and cooled down to 20°C by cold air circulation or natural cooling. It is then fed into a pulverizer for crushing and sieved through a mechanical screening device (through an 80-mesh sieve). The powder under the sieve is collected to obtain the compound probiotic type livestock and poultry growth-promoting feed additive.

[0094] Example 3:

[0095] This embodiment provides a compound probiotic-type growth-promoting feed additive for livestock and poultry and its preparation method, including the following steps:

[0096] Inject 150 parts by weight of deionized water into the reactor, turn on the reactor jacket cooling system, and control the water temperature inside the reactor at 30°C; start the high-shear disperser and adjust the shear rate to 5000 s. -1 25 parts by weight of the calcium-based bentonite were added at a uniform rate and run for 40 minutes to hydrate the bentonite particles. While maintaining the above shear rate, 2.0 parts by weight of the sodium hexametaphosphate were slowly added to the system within a 10-minute time window. High shear dispersion was continued for 30 minutes. After the viscosity of the system decreased due to thixotropy, the high shear device was turned off and the conventional paddle agitator was turned on. The speed was adjusted to 100 rpm to form a homogeneous suspension.

[0097] Adjust the flow rate of the coolant in the reactor jacket and strictly control the temperature of the material system inside the reactor at 25°C. Under low-speed stirring at 100 rpm, add 35 parts by weight of the D-trehalose dihydrate and 5 parts by weight of L-ascorbic acid sodium in sequence to the reactor and continue stirring for 20 minutes to dissolve them. Slowly and evenly sprinkle in 8 parts by weight of the sodium carboxymethyl cellulose powder and 4 parts by weight of gluconate-δ-lactone powder and maintain stirring for 50 minutes to ensure that the polymer materials are completely swollen. Pump in 50 parts by weight of the Lactobacillus plantarum fermentation concentrate obtained in Preparation Example 3, and continue mixing and stirring for 40 minutes to form a macroscopically homogeneous mixed slurry.

[0098] The resulting mixed slurry was transferred to a vacuum paddle dryer. The temperature of the jacket heating medium was controlled at 38°C. The vacuum pump was turned on, and the absolute pressure of the system was adjusted to maintain 12 kPa. The moisture was slowly evaporated under this condition. When the solid content of the material reached the predetermined range (i.e., when the solid content of the material reached 65%), the heating system was adjusted to raise the system temperature to 52°C at a heating rate of 2°C / min. At the same time, the vacuum valve was adjusted to reduce the absolute pressure of the system to 2.5 kPa. This temperature and pressure condition was maintained for 90 minutes. The system temperature was then raised to 60°C, and the absolute pressure was further reduced to 0.6 kPa. This condition was maintained until the residual moisture content of the material dropped to the predetermined range (i.e., dropped to 4%). Then the vacuum of the system was released to obtain the dried crude product.

[0099] The dried crude product is discharged from the equipment and cooled down to 25°C by cold air circulation or natural cooling. It is then fed into a pulverizer for crushing and sieved through a mechanical screening device (through a 100-mesh sieve). The powder under the sieve is collected to obtain the compound probiotic type livestock and poultry growth-promoting feed additive.

[0100] Comparative Examples 1-5:

[0101] Comparative Example 1:

[0102] Compared with Example 2, the difference is that sodium hexametaphosphate was removed from the raw material formula, but the rest are the same.

[0103] Comparative Example 2:

[0104] Compared with Example 2, the difference is that glucono-δ-lactone was removed from the raw material formula, but the rest are the same.

[0105] Comparative Example 3:

[0106] Compared with Example 2, the difference is that gluconate-δ-lactone was removed from the raw material formula, and when the temperature is raised to 48.5°C in the second stage of step 3, instead of relying on internal acid production, an equivalent amount of citric acid solution is pulsedly injected into the material through an external nozzle. All other aspects are the same.

[0107] Comparative Example 4:

[0108] Compared with Example 2, the difference is that in step two, the temperature of the material system in the reactor was mistakenly raised to 50°C and maintained, and then pumped into the dryer for drying in step three. All other aspects are the same.

[0109] Comparative Example 5:

[0110] Compared with Example 2, the difference lies in the change of excipients and preparation process. The Lactobacillus plantarum fermentation concentrate in Example 2 was mixed with an equal amount of trehalose, conventional dextrin and gelatin, and then directly subjected to conventional high-speed centrifugal spray drying (inlet air 130°C, outlet air 65°C). All other aspects were the same.

[0111] Test Examples 1-5:

[0112] Test Example 1: Rheological State Assessment during Pulping and Dispersion Stage

[0113] After mixing and stirring the Lactobacillus plantarum fermentation concentrate in the reactor to form a macroscopically homogeneous mixed slurry, the corresponding batch of mixed slurry is collected as the test sample, and the test sample is transferred to a sealed storage tank for standing to degas.

[0114] Turn on the rotary rheometer and install the coaxial cylindrical test rotor system. Adjust the Peltier temperature control frame of the instrument to keep the test environment temperature constant at 25°C.

[0115] Inject an appropriate amount of degassed slurry sample into the test cup, lower the test rotor to the specified gap, and scrape off any excess sample overflowing from the edges. Set a settling time to allow the internal temperature of the sample system to become uniform and for residual shear stress introduced during the sample loading process to dissipate.

[0116] Set up the steady-state shear test program, with the shear rate scan range set to 0.1 s. -1 up to 100s -1 The system uses a logarithmic distribution mode to collect data points and records the shear rate, apparent viscosity and shear stress data corresponding to each measurement point.

[0117] Set up a transient thixotropic test program and control the rotor shear rate from 0s. -1 The uniform acceleration is increased to 100s -1 After reaching the peak value, it decelerates to 0s with the same acceleration. -1 Record the acceleration curve (upward) and deceleration curve (downward), and perform area integration on the closed region enclosed by the two curves.

[0118] Table 1. Statistical table of apparent viscosity and thixotropic ring area of ​​each group of slurries at a specific shear rate.

[0119] Group <![CDATA[Test shear rate (s -1 )]]> Apparent viscosity (mPa·s) Thixotropic ring area (Pa / s) Example 2 50.0 3824.6 4512.8 Comparative Example 1 50.0 21453.7 31548.2 Comparative Example 4 50.0 17932.4 26739.5

[0120] in conclusion:

[0121] According to the data in Table 1, Example 2 was completed in 50 seconds. -1 The apparent viscosity at the shear rate was 3824.6 mPa·s, and the thixotropic ring area was 4512.8 Pa / s, indicating that the system exhibits thixotropy and the viscosity is within the design tolerance of the fluid pumping process. The apparent viscosity of Comparative Example 1 reached 21453.7 mPa·s, and that of Comparative Example 4 reached 17932.4 mPa·s, with both showing a several-fold increase in thixotropic ring area. In Comparative Example 1, which lacked sodium hexametaphosphate, calcium ions present in the interlayer of the calcium-based bentonite framework were exposed during shear dispersion, breaking free from their coordinated complexation and directly contacting the sodium carboxymethyl cellulose molecular chains in the feed system, resulting in early ionic cross-linking and causing the slurry to agglomerate and solidify during the physical mixing stage. In Comparative Example 4, the system temperature was raised to 50°C during the reactor mixing stage. This higher temperature triggered a ring-opening hydrolysis reaction of gluconate-δ-lactone, and the localized acidic environment caused the previously existing complex equilibrium to dissociate, releasing calcium ions that also triggered the construction of a macromolecular cross-linked network. A lack of space-shielding material or deviation from temperature control conditions during the pulping stage can cause the rheological parameters of the intermediate pulp to deviate from the processing requirements.

[0122] according to Figure 1According to the data, the shear stress in Example 2 increases gradually and non-linearly with the increase of shear rate. The curve starts near the origin of the coordinate system and increases when the shear rate reaches 100 s. -1 The shear stress only rose to about 280 Pa, and the curve did not produce a significant yield stress intercept on the vertical axis, reflecting the structural deentanglement and rearrangement process of macromolecular chain segments under the action of the shear flow field, and the fluid network system remained in a homogeneous suspension state. In contrast, the rheological test curves of Comparative Example 1 and Comparative Example 4 were generally shifted upward, producing initial stress intercepts of about 285 Pa and 230 Pa respectively at the zero shear point on the vertical axis, and at the end of the test interval (100 s). -1 The shear stress of the fluid all climbed to around 750 Pa, exhibiting typical plastic fluid characteristics.

[0123] The presence of this stress intercept of several hundred Pascals indicates that a three-dimensional spatial framework with specific strength has been pre-constructed within the material structure. Macroscopic flow requires an external mechanical force field to overcome this yield stress threshold. This premature solidification phenomenon deteriorates the rheological dynamics of the material, directly hindering the diffusion and uniform distribution of the subsequently added probiotic fermentation concentrate in the large-volume matrix. These test results demonstrate that introducing sodium hexametaphosphate into the formulation to construct a complexation shielding mechanism, combined with a temperature-controlled, acid-triggered mechanism within the reactor, can ensure effective material dispersion and operable rheological states in the early stages.

[0124] Test Example 2: In-situ Thermodynamic pH Response Test During Drying Process

[0125] In the preparation of compound probiotic-based growth-promoting feed additives for livestock and poultry, macroscopically homogeneous mixed slurries of each group are obtained and enter a vacuum paddle dryer. High-temperature resistant acid and alkali resistant online pH electrode probes and thermocouple temperature sensors are arranged in the core material zone inside the vacuum paddle dryer and connected to data acquisition terminal equipment to continuously record the temperature change trajectory and pH fluctuation of the material system in real time.

[0126] Considering that the online probe may experience poor local contact or measurement deviation due to the gradual solidification of the material, the sampling valve of the operating equipment is used for bypass quantitative sampling at the three key thermodynamic control nodes of the drying process: the end of the first stage slow evaporation period, the end of the second stage constant pressure maintenance period, and the end of the third stage extreme drying period.

[0127] Solid or semi-solid samples discharged from the sampling valve at each stage are rapidly placed in an ice-water bath for physical quenching to cut off any ongoing thermochemical reactions. Neutral deionized water is added to the sample according to a preset solid-liquid mass ratio, and the sample is thoroughly rehydrated using a mechanical homogenizer. The final pH value of the rehydrated suspension is measured using a benchtop high-precision pH meter calibrated with standard buffer solution, and the offline measurement data is compared and calibrated bidirectionally with the online sensor data at the corresponding time points.

[0128] The calibrated time, core temperature, and pH sequence data are imported into dedicated engineering calculation software to plot biaxial response curves of thermodynamic conditions and chemical environment, thereby monitoring and evaluating the actual operational efficiency of the thermally triggered acid-producing crosslinking mechanism.

[0129] Table 2. Record of pH values ​​within the system at the end of three different drying stages for each group of materials.

[0130] Group pH at the end of the first stage of slow evaporation The pH at the end of the second stage of isothermal and pressure maintenance period pH at the end of the third stage of extreme drying Example 2 6.782 4.651 4.314 Comparative Example 2 6.845 6.812 6.793 Comparative Example 3 6.804 5.426 5.385

[0131] in conclusion:

[0132] According to the data in Table 2, in Example 2, the system pH remained near neutral at 6.782 at the end of the first stage of slow evaporation, decreased to 4.651 at the end of the second stage of isothermal maintenance, and further decreased slightly to 4.314 at the end of the third stage. In Comparative Example 2, the system pH remained within a small range of 6.793 to 6.845 throughout the three test points of the drying process, without any substantial acidification reaction. In Comparative Example 3, after relying on external physical spraying of citric acid solution in the second stage, the overall pH measured after rehydration decreased to 5.426. Although this value deviated from the neutral point, it was far from reaching the acidity level established by the spontaneous acid production within Example 2.

[0133] The data trajectory of Example 2 verifies that gluconate-δ-lactone remains chemically inert in the low-temperature range during the initial drying stage, avoiding the destruction of the homogeneous dispersion of the liquid by early acid hydrolysis and cross-linking. As the drying process enters the set high-temperature period, gluconate-δ-lactone undergoes irreversible ring-opening hydrolysis, producing a large amount of gluconic acid, which gradually and completely transforms the internal environment of the system into an in-situ weakly acidic environment. In Comparative Example 2, due to the lack of endogenous acid-producing groups in the formulation, the alkaline medium environment of the system cannot be transformed, resulting in the inability to desorb and release calcium ions complexed with sodium hexametaphosphate. Although the external pulse acid addition operation in Comparative Example 3 macroscopically introduces acidic substances, the local concentration difference causes the instantaneous acidity of the material contact surface to be too high, triggering rapid cross-linking and crust formation of the polarized surface layer. This dense shell layer blocks the penetration and mass transfer of external acid solution into the core region of the material, resulting in an extremely limited overall homogenization pH decrease and a heterogeneous spatial distribution.

[0134] according to Figure 2The data showed that after drying for 60 minutes and entering the heating program, the core temperature curve of the system broke through the stable 34°C range and climbed to 48.5°C with the set process slope. Within the controlled temperature rise response range, the pH curve of Example 2, starting from 6.78, exhibited a smooth downward trajectory that mirrored the core temperature rise curve. This indicates that the ring-opening hydrolysis rate of gluconate-δ-lactone is strictly controlled by the thermodynamic temperature field of the system. When the temperature reaches the trigger threshold for molecular chemical bond breaking, the hydrolysis and acid production process occurs spontaneously and uniformly in all microscopic dimensions of the material, smoothly stripping calcium ions at the molecular scale and promoting in-situ homogeneous cross-linking of the system's macromolecular network.

[0135] The pH curve of Comparative Example 2 exhibits a horizontal extension completely unaffected by thermodynamic interference within the temperature rise and high-temperature maintenance range, indicating that the background material without external triggering possesses high-temperature stability. The pH curve of Comparative Example 3 shows an instantaneous step drop at the time of external pulsed spraying of citric acid, followed by a flattening at a high level accompanied by random measurement fluctuations, proving that the path relying on external physical spraying cannot establish a thermodynamically dynamic controlled slow-release acid production mechanism within the material. These in-situ thermodynamic monitoring results confirm that using gluconic acid-δ-lactone as a thermodynamic trigger in the formulation design can achieve precise temperature-pH unidirectional response control, thereby spontaneously completing the reconstruction of the homogeneous three-dimensional cross-linked network within the system at a specific drying phase change node.

[0136] Test Example 3: Matrix Compressive Yield Strength Test

[0137] Collect solid powder samples from each group after drying, pulverizing and sieving. Spread the powder material evenly into a wide-mouthed glass dish and place it in a constant temperature and humidity desiccator to stand and equilibrate for 24 hours.

[0138] Weigh a specified mass of equilibrium powder and load it into a cylindrical hard steel mold with an inner diameter of 10 mm. Operate the powder compression molding equipment to apply an axial mechanical static pressure of 10 MPa to the mold, and maintain this pressure for 60 seconds. Operate the demolding device to eject the formed cylindrical sample, and use vernier calipers to measure and record the actual height and cross-sectional diameter of the sample.

[0139] Turn on and calibrate the computer-controlled electronic universal testing machine. Place the prepared cylindrical specimen vertically in the center of the bottom pressure platform. Slowly lower the upper pressure plate, and set the current position as the zero point of the test displacement the instant the bottom surface of the upper pressure plate contacts the top of the specimen.

[0140] The universal testing machine was set to unidirectional constant-speed compression loading, and the displacement descent rate of the upper pressure plate was set to 1 mm per minute. A continuous testing command was initiated, and the testing machine system synchronously acquired the mechanical load feedback values ​​and corresponding displacement values ​​generated by the specimen during continuous compression deformation.

[0141] When the specimen structure fractures and disintegrates, causing a drop in real-time load force values, the compression program is terminated and the load-displacement data array recorded by the sensor is saved. Using material mechanics calculation formulas, the original data array is converted into engineering stress and strain values, the yield stress and fracture limit deformation point are extracted, and the total mechanical energy absorbed by the specimen before fracture is calculated by mathematical integration of the closed area of ​​the stress-strain curve.

[0142] Table 3. Statistical table of yield strength, elongation at break and compressive fracture energy of each group of specimens

[0143] Group Yield strength (MPa) Elongation at break (%) Compressive fracture energy (mJ) Example 1 15.24 12.63 148.53 Example 2 17.81 15.42 186.27 Example 3 16.53 14.18 165.71 Comparative Example 2 5.62 2.15 32.49 Comparative Example 3 8.94 3.87 56.84

[0144] in conclusion:

[0145] According to the data in Table 3, the yield strength of Examples 1 to 3 ranged from 15.24 MPa to 17.81 MPa, the elongation at break ranged from 12.63% to 15.42%, and the compressive fracture energy, reflecting the total energy absorption capacity of the samples, was as high as 148.53 mJ to 186.27 mJ. Comparative Example 2 had a yield strength of only 5.62 MPa, an elongation at break as low as 2.15%, and a compressive fracture energy of only 32.49 mJ. Comparative Example 3 had a yield strength of 8.94 MPa, an elongation at break of 3.87%, and a compressive fracture energy of 56.84 mJ. The mechanical properties of the Examples 1 to 3 were higher than those of the Comparative Example 2.

[0146] Comparative Example 2, lacking glucono-δ-lactone, lacked a spontaneous acidolysis triggering environment within its material system. No calcium ion stripping or macromolecular chain recombination reaction occurred, and the material matrix relied entirely on a single amorphous glassy phase formed after trehalose dehydration, leading to premature brittle fracture under external mechanical loads. Comparative Example 3, although applying citric acid solution to the surface via external spraying, resulted in localized cross-linking and crusting of the polarized surface layer, hindering further penetration. The internal core layer remained in a free state, and the overall deformation resistance and energy absorption capacity remained fundamentally unchanged. In the examples, glucono-δ-lactone induced hydrolysis and acid production during the drying thermodynamic stage, releasing the complexation shield of sodium hexametaphosphate. The released calcium ions and sodium carboxymethyl cellulose formed a homogeneous three-dimensional spatial network structure within the matrix. This cross-linked network provides geometric slip space at the macromolecular level, effectively absorbing external mechanical work and enhancing the matrix's resistance to compressive stress.

[0147] according to Figure 3The data from Comparative Examples 2 and 3 show that the stress-strain curves have a very steep upward slope and extremely narrow transverse deformation during the initial linear compressive deformation stage. After reaching the minimum ultimate stress, the bearing capacity drops vertically, and the curves do not show any gentle extension, reflecting the constitutive characteristics of low-energy brittle fracture. The stress-strain curves from Examples 1, 2, and 3 show obvious physical yielding after crossing the elastic deformation zone. A broad nonlinear plastic extension plateau appears at the top of the curve, with the stress value remaining high and accompanied by an increase in transverse strain. Finally, fracture occurs after exceeding the 12% deformation point.

[0148] The difference in the topological morphology of the curves confirms the transformation of the load-bearing mechanism of the material's mesoscopic structure. In the comparative glassy matrix, which has not undergone cross-linking, stress concentration easily occurs due to internal pore defects under external hydrostatic pressure, leading to rapid nucleation and penetrating propagation of microcracks, resulting in macroscopic disintegration failure. In the embodiment, the three-dimensional network structure formed by ion coordination can effectively transfer and dissipate externally applied mechanical compressive and shear stresses. At the crack initiation endpoint, the fracture energy is consumed through conformational unentanglement and rearrangement of molecular chains, inhibiting the evolution of microscopic defects into macroscopic fracture surfaces. This internal toughening mechanism fundamentally optimizes the compressive mechanical properties of the brittle glassy matrix, enabling the powdered granular matrix encapsulating the fermentation concentrate to acquire the physical toughness to withstand high-strength granulation and extrusion processes.

[0149] Test Example 4: Probiotic Survival Rate Test under Extreme Industrial Granulation Conditions

[0150] Standard pig feed powder was used as the carrier matrix, and samples were taken for background microbial testing.

[0151] Weigh out each group of probiotic feed additive powders, add them to the standard pig feed powder according to the ratio, put them into the twin-shaft paddle mixer, and start the equipment until the materials are mixed.

[0152] After shutdown, multiple random samples were taken from different areas inside the mixer. The mixed powder samples were weighed, serially diluted with sterile physiological saline, and inoculated onto MRS agar plates. The petri dishes were transferred to an anaerobic incubator at 37°C and incubated for 48 hours. Colony counts were performed, and the initial viable count of each mixed powder group before granulation was recorded.

[0153] The mixture is continuously fed into the industrial ring die pellet mill. The steam pipeline valves are adjusted to maintain the conditioning temperature of the material in the conditioner at 85°C, and the conditioning process is maintained for 45 seconds. The operating parameters of the ring die are set to ensure that the mechanical compressive stress experienced by the material as it is extruded through the die holes reaches 4 MPa. The discharged pellets are collected at the outlet.

[0154] The formed pelleted feed was transferred to a counter-flow cooler for ventilation and cooling. After the internal temperature of the material dropped to the room temperature range, it was sent to a pulverizer for mechanical pulverization. The pulverized powder sample was weighed, and the viable bacteria count after the pelleting process was determined according to the gradient dilution and plate coating method described above. The percentage of viable bacteria survival rate and the logarithmic decrease value of each group of materials before and after pelleting were calculated.

[0155] Table 4. Comparison of viable bacteria count and survival rate percentage of each group of feed additives before and after pelleting.

[0156] Group viable cell count before granulation (CFU / g) viable cell count after granulation (CFU / g) Survival rate (%) Log decrease value Example 1 <![CDATA[4.85×10 7 ]]> <![CDATA[3.12×10 7 ]]> 64.33 0.19 Example 2 <![CDATA[5.42×10 7 ]]> <![CDATA[4.15×10 7 ]]> 76.57 0.12 Example 3 <![CDATA[6.03×10 7 ]]> <![CDATA[4.28×10 7 ]]> 70.98 0.15 Comparative Example 1 <![CDATA[5.18×10 7 ]]> <![CDATA[3.65×10 6 ]]> 7.05 1.15 Comparative Example 2 <![CDATA[5.76×10 7 ]]> <![CDATA[8.92×10 5 ]]> 1.55 1.81 Comparative Example 3 <![CDATA[5.34×10 7 ]]> <![CDATA[1.58×10 6 ]]> 2.96 1.53 Comparative Example 4 <![CDATA[4.95×10 7 ]]> <![CDATA[4.12×10 6 ]]> 8.32 1.08 Comparative Example 5 <![CDATA[5.51×10 7 ]]> <![CDATA[2.36×10 4 ]]> 0.04 3.37

[0157] in conclusion:

[0158] According to the data in Table 4, after undergoing conditioning at 85℃ and ring die extrusion granulation at 4MPa, the measured viable cell count in Examples 1 to 3 was 3.12 × 10⁻⁶. 7 CFU / g up to 4.28×10 7 The viable cell viability (CFU / g) remained between 64.33% and 76.57%, with corresponding Log decrease values ​​ranging from 0.12 to 0.19. Comparative Example 5, after undergoing the same granulation process, showed a viable cell viability of 0.04%, with a Log decrease value of 3.37. Comparative Examples 2 and 3 showed viable cell viability of 1.55% and 2.96%, respectively, with Log decrease values ​​of 1.81 and 1.53. Comparative Examples 1 and 4 showed viability of 7.05% and 8.32%, respectively, with Log decrease values ​​of 1.15 and 1.08.

[0159] according to Figure 4 The data, specifically the height of the bars and the distribution of discrete points in the bar chart, reflect the survival status of viable bacteria in each group of materials under pressure and heat conditions. The attenuation index value of Comparative Example 5 is located in the high region of the chart's vertical axis. The formulation system of Comparative Example 2 relies on an amorphous trehalose matrix, which undergoes brittle fracture during mechanical loading, exposing the internally encapsulated fermentation concentrate. High-temperature conditioning steam leads to the inactivation of the exposed microbial cells. In Comparative Example 3, the application of exogenous acid causes the material surface to form an isolated cross-linked structure. The shear force generated during the ring die extrusion stage destroys the surface hard shell, failing to provide global support to the particle core, resulting in the disintegration of internal free matter.

[0160] The examples demonstrate a macroscopic composite matrix formed by a thermodynamically triggered in-situ homogeneous cross-linked network and rigid bentonite particles. Under 4 MPa compressive stress, this matrix exhibits deformation redundancy characteristics of the network structure, reducing the probability of microcrack propagation. The dense cross-linked architecture limits the migration rate of external moisture and heat to the core fermentation concentrate region, reducing heat accumulation. In Comparative Example 1, the lack of sodium hexametaphosphate ion complexation resulted in heterogeneous agglomerates within the reaction system, causing uneven spatial distribution and varying encapsulation thickness of probiotics. Bacteria in the outer layer and weakly encapsulated areas died during the granulation process. In Comparative Example 4, misoperation caused premature cross-linking of the polymer system, forming a high-viscosity gel block before drying and dehydration. This interfered with the structural uniformity of subsequent drying and granulation, leading to a decrease in physical properties resistant to external damage.

[0161] Test Example 5: Long-term Stability Test at Room Temperature

[0162] Take the powder products obtained by final drying in Example 2 and Comparative Example 5, weigh equal amounts of the samples, and put them into three-layer composite packaging bags made of polyester, aluminum foil and polyethylene. Use a heat-sealing machine to close and seal the bag openings. No additional vacuuming or nitrogen filling is performed during the sealing process.

[0163] A fixed mass of the unpackaged original powder sample from month 0 was weighed and added to sterile physiological saline. The mixture was then vortexed until the powder was completely dispersed. The dispersion was serially diluted tenfold with sterile physiological saline. Specified volumes of each dilution were spread onto MRS solid agar plates and incubated at 37°C for 48 hours. Colony-forming units on the plates were counted, and the initial absolute viable count for each group at month 0 was recorded.

[0164] Place the sealed packaging bags containing the samples flat on the shelves of the artificial climate constant temperature and humidity test chamber. Set the internal operating temperature of the chamber to 25℃ and the relative humidity to a stable 70%, then start the equipment for continuous room temperature accelerated aging storage.

[0165] At the milestones of 1 month, 3 months, and 6 months of continuous operation of the equipment, the corresponding batches of test packaging bags were removed from the test chamber. The bags were opened, and the powder samples after aging treatment inside the chamber were weighed. The absolute viable count of each group of samples was determined at the end of different storage periods using the same gradient dilution and anaerobic coating culture method as described above.

[0166] Summarize the plate colony count data for all testing periods. Divide the absolute viable count values ​​for month 1, month 3, and month 6 by the initial viable count value for month 0 of the corresponding group, and calculate and record the viable retention rate at each time point.

[0167] Table 5. Absolute viable count tracking table for each group of samples at different storage periods.

[0168] Group 0 months (CFU / g) 1 month (CFU / g) 3 months (CFU / g) 6 months (CFU / g) Example 2 <![CDATA[5.41×10 7 ]]> <![CDATA[4.98×10 7 ]]> <![CDATA[4.15×10 7 ]]> <![CDATA[3.46×10 7 ]]> Comparative Example 5 <![CDATA[5.52×10 7 ]]> <![CDATA[1.63×10 7 ]]> <![CDATA[5.84×10 5 ]]> <![CDATA[1.97×10 4 ]]>

[0169] in conclusion:

[0170] According to the data in Table 5, in Example 2, during the 6-month storage period at 25°C and 70% relative humidity, the measured viable count increased from the initial 5.41 × 10⁻⁶. 7 CFU / g decreased to 3.46×10 7 CFU / g. In Comparative Example 5, under the same environmental parameters, the measured viable count increased from the initial 5.52 × 10⁻⁶. 7 CFU / g decreased to 1.97×10 4 CFU / g.

[0171] according to Figure 5 The data, with the shaded area representing the error band, shows that the viable bacteria retention rate curve of Example 2 exhibits a gradual decline throughout the testing period, maintaining a retention rate of 63.95% at the 6th month testing node. In contrast, the retention rate curve of Comparative Example 5 drops sharply to 29.52% at the 1st month testing node, subsequently exhibiting a non-linear and rapid decline, reaching a near-zero level at the 6th month. The decline range of the curves reflects the difference in the barrier effectiveness of the microscopic embedding medium against external environmental factors.

[0172] Example 2 utilizes calcium-based bentonite particles hydrated under high pressure to construct a rigid silicate support framework. Combined with the amorphous glass phase formed during the vacuum thermal dehydration of trehalose, a dense, continuous physical barrier without regular grain boundaries is assembled at the mesoscale. This rigid composite structure interrupts the capillary permeation pathway of environmental water molecules and blocks the diffusion channels of free oxygen atoms. Because the cross-linked polymer network triggered by the in-situ acidification process is interwoven and stabilized between the glass phase and silicate particles, the matrix structure's resistance to plastic deformation is greatly enhanced, inhibiting the relaxation movement of polymer chains under high humidity conditions and preventing the reverse transformation of the amorphous glass phase to a rubber phase. The *Lactobacillus plantarum* within the encapsulated core is protected from intracellular lipid peroxidation caused by oxygen leakage and proteolytic hydrolysis caused by water release.

[0173] Comparative Example 5 used a conventional gelatin and dextrin blend as the wall material and underwent high-speed centrifugal spray drying. The porosity of the outer surface of the molded embedded particles was too large, resulting in inherent defects in the continuity of the outer film. In an accelerated aging environment with 70% relative humidity, the porosity triggered a strong hygroscopic and rehydration effect, causing the outer matrix to soften and collapse over a large area due to water absorption. This resulted in the loss of its physical barrier function against oxygen and water molecules, leading to a logarithmic inactivation and decay of the core viable bacterial community inside.

Claims

1. A composite probiotic type of growth promoting feed additive for livestock and poultry characterized in that, Made from the following ingredients in parts by weight: 15-25 parts of calcium-based bentonite; Sodium hexametaphosphate 0.5–2.0 parts; 20-35 parts of D-trehalose dihydrate; 2-5 parts of L-ascorbic acid sodium; 3-8 parts of sodium carboxymethyl cellulose powder; 1-4 parts of glucono-δ-lactone powder; 30-50 parts of Lactobacillus plantarum fermentation concentrate; The live bacterial count concentration of the Lactobacillus plantarum fermentation concentrate is 1 x 10 10 ~ 5 x 10 10 CFU / mL.

2. The composite probiotic type growth promoting feed additive for poultry and livestock as claimed in claim 1 wherein, The preparation method of the Lactobacillus plantarum fermentation concentrate includes: Lactobacillus plantarum strains were inoculated into modified MRS liquid medium and subjected to isothermal anaerobic fermentation until the end of the logarithmic growth phase to obtain the fermentation broth. The fermentation broth was pumped into a centrifuge for centrifugation, the supernatant was discarded, and the bottom bacterial sludge was collected. The bottom layer of bacterial sludge was resuspended using sterile physiological saline to obtain a concentrated fermentation solution of Lactobacillus plantarum.

3. The composite probiotic growth promoting feed additive for poultry and livestock as claimed in claim 2, wherein, When preparing the Lactobacillus plantarum fermentation concentrate, the fermentation tank temperature is set at 35-37°C, the initial pH value is controlled at 6.0-6.5, the constant temperature anaerobic fermentation time is 16-24 hours, the centrifugation speed is 4000-6000 rpm, and the centrifugation time is 10-15 minutes.

4. A process for the preparation of a composite probiotic type growth promoting feed additive for poultry and livestock characterized in that, The preparation of the compound probiotic-type livestock and poultry growth-promoting feed additive according to any one of claims 1 to 3 includes the following steps: Inject deionized water into the reactor and control the water temperature inside the reactor; start the high shear disperser and add calcium-based bentonite at a uniform speed; maintain the above shear rate, add sodium hexametaphosphate into the system, continue high shear dispersion, turn off the high shear disperser, turn on the conventional paddle agitator, and form a homogeneous suspension. Strictly control the temperature of the material system inside the reactor. Under low-speed stirring, add the D-trehalose dihydrate and L-ascorbic acid sodium salt to the reactor in sequence and continue stirring. Slowly and evenly sprinkle in sodium carboxymethyl cellulose powder and gluconate-δ-lactone powder while maintaining stirring. Pump in Lactobacillus plantarum fermentation concentrate and continue mixing and stirring to form a macroscopically homogeneous mixed slurry. The resulting mixed slurry is transferred to a vacuum paddle dryer. The temperature of the jacket heating medium is controlled, the vacuum pump is turned on, and the absolute pressure of the system is adjusted to maintain this state and slowly evaporate the moisture. When the solid content of the material reaches the predetermined range, the heating system is adjusted to raise the system temperature, and the vacuum valve is adjusted simultaneously to lower the absolute pressure of the system. This temperature and pressure condition is maintained constant. The system temperature is further increased, and the absolute pressure is further reduced. This state is maintained until the residual moisture content of the material drops to the predetermined range. Then the system vacuum is released to obtain the dried crude product. The dried crude product is discharged from the equipment and cooled by cold air circulation or natural cooling. It is then fed into a pulverizer for crushing and sieved through a mechanical screening device. The undersized powder is collected to obtain the compound probiotic type livestock and poultry growth-promoting feed additive.

5. The preparation method according to claim 4, characterized in that, The amount of deionized water injected is 100-150 parts by weight, and the water temperature inside the reactor is controlled at 20-30℃; the shear rate is adjusted to 3000-5000 s. -1 The running time after adding the calcium-based bentonite is 20-40 minutes; the addition time window for the sodium hexametaphosphate is 5-10 minutes, and the high-shear dispersion time is 15-30 minutes.

6. The preparation method according to claim 4, characterized in that, Strictly control the temperature of the material system inside the reactor to 20-25℃; adjust the speed of the conventional paddle agitator to 50-100 rpm; continue stirring for 15-20 minutes after adding the D-trehalose dihydrate and L-ascorbic acid sodium; maintain stirring for 30-50 minutes after sprinkling in the sodium carboxymethyl cellulose powder and glucono-δ-lactone powder; and continue mixing and stirring for 20-40 minutes after pumping in the Lactobacillus plantarum fermentation concentrate.

7. The preparation method according to claim 4, characterized in that, After the mixed slurry is transferred to the vacuum paddle dryer, the temperature of the jacket heating medium is controlled at 30-38°C, and the absolute pressure of the system is maintained at 8-12 kPa; the time when the solid content of the material reaches the predetermined range refers to when the solid content of the material reaches 55%-65%.

8. The preparation method according to claim 7, characterized in that, When the solid content of the material reaches 55%–65%, the system temperature is increased to 45–52°C at a heating rate of 1–2°C / min, and the absolute pressure of the system is reduced to 1.5–2.5 kPa. This temperature and pressure condition is maintained constant for 40–90 minutes. The system temperature is then further increased to 53–60°C, and the absolute pressure is further reduced to 0.1–0.6 kPa. The phrase "until the residual moisture content of the material drops to a predetermined range" means until the residual moisture content of the material drops to 4%–6%.

9. The preparation method according to claim 4, characterized in that, The material is cooled down to 15-25°C.

10. The method of claim 9, wherein, The sieving process described above refers to passing the material through a 60-100 mesh sieve.

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