Fermented feed additive for improving intestinal health of piglets and preparation method thereof

Abstract

The invention relates to the technical field of biological feed additives, and discloses a fermented feed additive for improving intestinal health of piglets and a preparation method thereof, and the additive is prepared from a solid state fermentation substrate, sodium alginate, chitosan, beta-tricalcium phosphate and a lactobacillus plantarum seed solution. In the fermentation process, lactobacillus plantarum is utilized to produce acid through metabolism to induce beta-tricalcium phosphate to be dissolved and release calcium ions, and a sodium alginate-chitosan interpenetrating polymer network gel structure is formed through in-situ crosslinking. The preparation method comprises the steps of solid precursor mixing, inoculation and tempering, metabolism triggering type standing fermentation and freeze drying. Synchronous embedding of thalli and metabolites is achieved through in-situ gelation, the obtained product has pH responsiveness, gastric acid erosion can be effectively blocked, fixed-point release of intestinal tracts is achieved, and high viable bacterium concentration is maintained.

Description

Technical Field

[0001] This invention relates to the field of biological feed additives, and in particular to a fermented feed additive for improving the intestinal health of piglets and its preparation method. Background Technology

[0002] Lactobacillus plantarum, an important probiotic, is widely used in the feed of weaned piglets to regulate intestinal flora balance, inhibit the growth of pathogens, and improve immunity. Solid-state fermentation is a common process for preparing such additives, transforming feed matrices such as soybean meal and wheat bran into biological feed rich in active probiotics, small molecule peptides, and organic acids through microbial fermentation.

[0003] However, conventional solid-state fermentation technology suffers from low viable cell survival rates and poor targeted release capabilities in practical applications. During fermentation, as *Lactobacillus plantarum* undergoes metabolic activity, large amounts of lactic acid and other organic acids accumulate in the fermentation system, leading to a significant decrease in pH. This continuously intensifying acidic environment stresses the bacteria themselves, resulting in a substantial decline in viable cell count during the later stages of fermentation and storage. Furthermore, the gastric juice of piglets is highly acidic; *Lactobacillus plantarum* without effective protection is easily killed by acid as it passes through the stomach, making it difficult to reach its intestinal colonization site with high activity and limiting its probiotic functions.

[0004] To improve the stress resistance of probiotics, current technologies often employ microencapsulation, which involves first obtaining a high concentration of bacterial powder, then mixing it with wall materials such as sodium alginate and starch, followed by spray drying or extrusion granulation. This step-by-step process of "fermentation first, then encapsulation" has limitations: on the one hand, the thermal effects and mechanical shear forces during drying and granulation can cause secondary damage to heat-sensitive probiotics; on the other hand, this physical encapsulation method is complex, and the bonding force between the wall material and the fermentation substrate is weak, making it prone to breakage during feed mixing and transportation.

[0005] Some studies have attempted to construct a gel protective layer in feed using the cross-linking reaction between sodium alginate and calcium ions. However, in conventional operations, soluble calcium salts such as calcium chloride are usually added directly to the system. Because the reaction rate between calcium ions and sodium alginate is extremely fast, direct contact leads to instantaneous gelation at the contact surface, forming local agglomerates or crusts, preventing the formation of a uniform encapsulation network in the fermentation matrix. Furthermore, vigorous mechanical stirring for dispersion destroys the already formed gel structure, significantly reducing its barrier properties against gastric acid. Therefore, achieving gentle, uniform, and structurally intact in-situ encapsulation in solid-state fermentation systems to simultaneously address fermentation acid inhibition and low gastric survival rates is a pressing technical challenge. Summary of the Invention

[0006] The technical problem solved by this invention is that the existing Lactobacillus plantarum feed additives lack an effective encapsulation and protection mechanism during solid-state fermentation, which makes the bacteria susceptible to high concentrations of acid stress and inactivation in the later stage of fermentation. Furthermore, the products produced have a low survival rate when passing through the highly acidic environment of piglets' stomachs and are difficult to achieve targeted release in the intestines.

[0007] To address the above problems, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a fermented feed additive for improving the intestinal health of piglets. This additive is a gel-state fermented product with an interpenetrating polymer network structure, made from raw materials comprising the following parts by weight: 95-105 parts solid fermentation substrate; 3-8 parts sodium alginate; 1-4 parts chitosan; 2-5 parts tricalcium β-phosphate; and 2-5 parts *Lactobacillus plantarum* seed culture; wherein the viable cell concentration of the *Lactobacillus plantarum* seed culture is 2.0 × 10⁻⁶. 9 CFU / mL ~ 4.0 × 10 9 CFU / mL.

[0009] Sodium alginate, chitosan, and β-tricalcium phosphate serve as gel precursors. During fermentation, β-tricalcium phosphate releases calcium and phosphate ions through acid production via Lactobacillus plantarum metabolism, which then crosslinks in situ to form an interpenetrating polymer network gel structure containing Lactobacillus plantarum and its metabolites.

[0010] By employing the above technical solution, this invention utilizes the chemical correlation between microbial metabolic acid production and the phase transition of the gel precursor to achieve simultaneous encapsulation of bacterial cells and metabolites. The specific reaction mechanism is as follows:

[0011] 1. Establish a metabolic acid-induced delayed ion release system:

[0012] This scheme uses β-tricalcium phosphate as a pH-sensitive calcium source release agent. In the early stage of fermentation (neutral or weakly acidic environment), β-tricalcium phosphate remains solid and insoluble, avoiding the instantaneous local gelation caused by traditional free calcium ions (such as calcium chloride), ensuring that Lactobacillus plantarum is fully dispersed and proliferates in a low-viscosity matrix.

[0013] 2. In-situ construction of interpenetrating polymer network (IPN) structures:

[0014] As *Lactobacillus plantarum* accumulates lactic acid through metabolism, the following physicochemical processes occur when the pH of the system drops to the solubility threshold (pH < 5.5):

[0015] Calcium-sodium alginate network formation: The acidic environment promotes the dissolution of β-tricalcium phosphate and the release of calcium ions (Ca). 2+ ), Ca 2+It undergoes a coordination chelation reaction with the guluronic acid units in the sodium alginate molecular chain to form a primary gel backbone, thereby fixing the bacterial cells in situ.

[0016] Polyelectrolyte network composite: Under acidic conditions, chitosan aminoprotonation (-NH3) + The positively charged sodium alginate carboxyl group (-COO) is similar to the negatively charged sodium alginate carboxyl group. - The electrostatic interaction between the phosphate ions and the electrostatic composite network and the aforementioned calcium-sodium alginate chemical cross-linking network forms a dense interpenetrating network structure.

[0017] 3. pH-responsive barrier and release characteristics:

[0018] The resulting IPN gel structure exhibits significant pH responsiveness. In a simulated gastric juice environment (pH 2.0), sodium alginate carboxylation leads to network contraction, and the dense gel layer hinders hydrogen ion penetration, maintaining a relatively stable internal microenvironment. In the intestinal environment (pH > 6.0), due to enhanced electrostatic repulsion and calcium ion exchange, the gel network swells and gradually dissociates, releasing the embedded Lactobacillus plantarum.

[0019] Preferably, the raw materials consist of the following components in parts by weight: 45-60 parts soybean meal, 25-30 parts corn flour, 15-30 parts wheat bran; 5-6 parts sodium alginate; 2.5-3 parts chitosan; 3-4 parts tricalcium β-phosphate; and 3-4 parts Lactobacillus plantarum seed liquid.

[0020] By adopting the above technical solution, the ratio of soybean meal, corn flour and wheat bran balances the supply of carbon and nitrogen sources, regulates the acid production kinetics of Lactobacillus plantarum, and matches it with the gelation rate, avoiding local uneven gelation due to excessively rapid acid production or insufficient gel strength due to excessively slow acid production.

[0021] Preferably, the sodium alginate has a mannulic acid / guluronic acid (M / G) ratio of 0.5 to 1.0, and its 1% aqueous solution has an apparent viscosity of 100 to 300 mPa·s at 20°C; the chitosan has a degree of deacetylation ≥ 85% and a weight-average molecular weight of 50 to 150 kDa.

[0022] By adopting the above technical solutions, the M / G ratio and viscosity range of sodium alginate are limited to ensure that it combines with calcium ions to form a gel framework with suitable mechanical strength; the degree of deacetylation and molecular weight of chitosan are limited to ensure that it has sufficient charge density in an acidic environment to enhance electrostatic recombination and improve the stability of the gel structure in a low pH environment.

[0023] Preferably, the β-tricalcium phosphate begins to dissolve and release calcium ions and phosphate buffer pairs when the pH of the fermentation system drops below 5.5, maintaining the pH value between 4.0 and 4.6 in the later stage of fermentation.

[0024] By employing the above technical solution, β-tricalcium phosphate acts as both a cross-linking agent and a pH buffer. The phosphate components produced by its dissolution construct a buffer system, inhibiting excessive pH drop in the later stages of fermentation and preventing irreversible damage to *Lactobacillus plantarum* due to excessive acidity, thereby maintaining a high viable cell count at the fermentation endpoint.

[0025] Preferably, the viable bacteria survival rate of the fermented feed additive is ≥75% after being treated in simulated gastric juice at pH 2.0 for 2 hours, and the lactic acid release concentration in the liquid phase of the treated simulated gastric juice is ≤300mg / L.

[0026] By employing the above technical solution, the aforementioned physical parameters characterize the barrier efficacy of the IPN gel structure. A high survival rate indicates that the gel layer effectively blocks gastric acid erosion; a low lactic acid release indicates that the gel matrix achieves effective loading of metabolites, reducing the loss of active ingredients before reaching the intestinal target.

[0027] Secondly, the present invention provides a method for preparing a fermented feed additive to improve the intestinal health of piglets, comprising the following steps:

[0028] S1. Solid precursor mixing: Sodium alginate, chitosan and β-tricalcium phosphate are premixed evenly and then mixed with the pulverized solid fermentation substrate to obtain solid mixture.

[0029] S2. Inoculation and conditioning: Disperse the Lactobacillus plantarum seed liquid in sterile water, add it to the solid mixture in the form of a spray, and adjust the initial pH to 6.5-7.0;

[0030] S3, Metabolic Triggered In-Situ Gel Fermentation: The material obtained in S2 is sealed and allowed to ferment statically at 30-37℃ for 24-48 hours. No mechanical stirring is performed during the fermentation process. In-situ gelation is initiated by metabolic acid production.

[0031] S4. Post-processing: After fermentation, the gel-like material is dried and pulverized to obtain the finished product.

[0032] By adopting the above technical solution, this invention achieves simultaneous fermentation and propagation processes and gel formation processes. The specific process principle is as follows:

[0033] 1. Homogeneous gelation mechanism based on in-situ acid production:

[0034] This method utilizes *Lactobacillus plantarum* dispersed in a matrix to produce lactic acid, establishing an in-situ acidic environment. Unlike traditional methods that involve adding exogenous cross-linking agents (such as calcium chloride solution), which can easily lead to excessively rapid local reactions or surface crusting, this method uses *Lactobacillus plantarum* as uniformly distributed acid-producing sites, promoting the diffusion of hydrogen ions from the matrix interior to the exterior, inducing the dissolution of adjacent β-tricalcium phosphate particles. This inside-out triggering mechanism ensures the spatial uniformity of calcium ion release, thereby forming a structurally homogeneous gel matrix.

[0035] 2. Protection of gel network structure during static fermentation:

[0036] During the critical stage of the sol-gel transition, the chemical crosslinks (calcium-sodium alginate) and electrostatic composites (chitosan-sodium alginate) formed within the system exhibit weak bonding forces and are sensitive to shear stress. This method employs a static fermentation process in step S3, strictly excluding mechanical stirring to prevent shear stress from damaging the nascent gel network. This ensures the complete construction of the interpenetrating polymer network (IPN), ultimately resulting in a macroscopically elastic gel mass.

[0037] 3. Simplify the process and reduce cell damage:

[0038] This method combines the three processes of microbial proliferation, metabolite accumulation, and carrier encapsulation into a single step. This simultaneous process reduces material transfer and post-processing steps, lowering the risk of contamination. Furthermore, since gel encapsulation mainly occurs in the later stages of fermentation, it effectively reduces mechanical damage and thermal stress to the microorganisms during subsequent drying and pulverization processes.

[0039] Preferably, the solid fermentation substrate includes soybean meal, corn flour and wheat bran, and the mixing uniformity CV after mixing is ≤5%; in step S2, the amount of sterile water added is such that the material-to-water ratio is controlled between 1:0.8 and 1:1.2.

[0040] By adopting the above technical solutions, controlling the mixing uniformity ensures the uniform spatial distribution of the gel precursor and prevents differences in gel strength caused by local concentration gradients; controlling the material-to-water ratio within a specific range provides the necessary liquid-phase mass transfer environment for microbial metabolism and ensures that the system has a suitable solid content, so that the formed gel skeleton has sufficient mechanical support.

[0041] Preferably, in step S2, the initial pH is adjusted using NaOH solution to ensure that the initial environment is suitable for the growth of Lactobacillus plantarum and does not trigger the acidic gelation of sodium alginate; in step S3, the pH of the system is controlled between 4.0 and 4.2 at the end of fermentation.

[0042] By adopting the above technical solution, adjusting the initial pH value avoids premature gelation caused by local acidic areas and shortens the lag period of the bacteria; controlling the final pH value at 4.0 to 4.2 achieves a balance between gel strength and bacterial activity: this pH range can ensure that β-tricalcium phosphate is fully dissolved to provide sufficient cross-linking density, and can also avoid excessive acidity causing survival pressure on Lactobacillus plantarum.

[0043] Preferably, in step S3 of the preparation method, the release of calcium ions and pH buffering are achieved simultaneously through the dissolution of β-tricalcium phosphate, so that the storage modulus of the fermentation system reaches more than 150 Pa at the end of fermentation, and the storage modulus is greater than the loss modulus.

[0044] By adopting the above technical solution, the relationship and numerical change between the storage modulus (G') and the loss modulus (G'') reflect the rheological state of the system. When G' is greater than 150 Pa and G' is greater than G'', it indicates that the system has completed the phase transition from a viscous fluid to an elastic solid gel. This physical index can be used as a basis for judging the completion of the in-situ gelation reaction and the maturity of the network structure construction.

[0045] Preferably, in step S4, the drying process employs a vacuum freeze-drying process with the following parameters: pre-freezing temperature below -30°C, vacuum drying pressure ≤15Pa, desorption drying temperature ≤25°C, and drying to a moisture content ≤10%.

[0046] By adopting the above technical solution, water is removed by sublimation under low temperature and low pressure, preventing the collapse of the gel network caused by high temperature, and preserving the porous structure of the wet gel and the biological activity of Lactobacillus plantarum to the maximum extent.

[0047] In summary, the present invention has at least one of the following beneficial technical effects:

[0048] 1. This invention induces the in-situ formation of a sodium alginate-chitosan-calcium interpenetrating polymer network (IPN) gel structure through acid production from Lactobacillus plantarum metabolism. This structure utilizes the acid-induced shrinkage of sodium alginate and the electrostatic recombination of chitosan to form a dense barrier layer in a strongly acidic environment simulating gastric juice. This effectively blocks the diffusion of hydrogen ions into the gel interior, significantly improving the survival rate of Lactobacillus plantarum in the acidic gastric environment and enabling subsequent responsive disintegration and release in the intestinal environment.

[0049] 2. This invention utilizes β-tricalcium phosphate as a pH-responsive crosslinking agent and buffer. The phosphate component produced by the dissolution of β-tricalcium phosphate under acidic conditions constructs an in-situ buffer system, maintaining the pH value within a suitable range during the later stages of fermentation and avoiding acid damage to *Lactobacillus plantarum* caused by an excessively acidic environment. Simultaneously, its delayed dissolution characteristics ensure uniform release of calcium ions, preventing uneven gel structure caused by excessively rapid local reactions and increasing the viable cell concentration at the fermentation endpoint.

[0050] 3. This invention employs a metabolically triggered static fermentation process, avoiding the damage to the nascent gel network caused by the shear force generated by mechanical stirring. This static molding method ensures the integrity of the interpenetrating polymer network structure, giving the final product a high storage modulus and mechanical strength. Combined with a vacuum freeze-drying process, it achieves effective loading and long-term preservation of heat-sensitive probiotics and metabolites, solving the problem of poor storage tolerance in traditional fermented feed additives. Attached Figure Description

[0051] Figure 1 This is a comparison chart of monitoring data on the changes of key parameters over time during the fermentation process of Examples 1, 3, and 6 in Test Example 1 of the present invention; wherein, a) is a comparison of the pH value change trend during the 48-hour fermentation process of Examples 1, 3, and 6; b) is a comparison of the storage modulus change trend over time during the fermentation process of Examples 1, 3, and 6; c) is a comparison of the loss modulus change trend over time during the fermentation process of Examples 1, 3, and 6; d) is a numerical comparison of the storage modulus and loss modulus during the fermentation process of Example 1; e) is a numerical comparison of the storage modulus and loss modulus during the fermentation process of Comparative Example 3; and f) is a numerical comparison of the storage modulus and loss modulus during the fermentation process of Comparative Example 6. Detailed Implementation

[0052] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only for explaining this invention and are not intended to limit the scope of protection of this invention.

[0053] 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.

[0054] Soybean meal, corn flour, and wheat bran are all commercially available feed-grade products, with soybean meal containing ≥43% crude protein and corn flour containing ≥65% starch.

[0055] Sodium alginate, CAS No.: 9005-38-3, food grade, its 1% (w / v) aqueous solution has an apparent viscosity of 100-300 mPa·s at 20℃, and a mannulic acid / guluronic acid (M / G) ratio of 0.5-1.0.

[0056] Chitosan, CAS No.: 9012-76-4, food grade, degree of deacetylation (DD) ≥ 85%, weight average molecular weight (Mw) 50-150 kDa, powder particle size passing through 100 mesh sieve.

[0057] Lactobacillus plantarum, strain preservation number ATCC 14917, its seed culture preparation is shown in the preparation example below.

[0058] Preparation Example 1:

[0059] This preparation example provides a Lactobacillus plantarum seed solution for fermentation inoculation, comprising the following steps:

[0060] (1) Activation of bacterial strain: Lactobacillus plantarum was streaked onto MRS agar plates under aseptic conditions and placed in a 37°C constant temperature incubator and inverted for 24 hours until plump, milky white single colonies with neat edges grew.

[0061] (2) Preparation of primary seed culture: Pick a single colony from the plate and inoculate it into an Erlenmeyer flask containing 50 mL of sterile MRS liquid culture medium. Incubate at 37°C in a static state or at a low speed (50-80 rpm) for 18 hours to obtain primary seed culture.

[0062] (3) Secondary seed culture expansion: Transfer the primary seed culture at an inoculation rate of 2% (v / v) to a seed tank or large conical flask containing 1L of sterile MRS liquid culture medium, and incubate at 37℃ or at low speed (50-80 rpm) for 16-20 hours.

[0063] (4) Quality inspection and collection: Microscopic examination revealed that the bacterial cells were short rod-shaped and free from contamination by other bacteria. The OD of the bacterial solution was measured. 600 The values ​​were then analyzed and plate counts were performed. The final viable cell concentration of *Lactobacillus plantarum* seed culture was 2.0 × 10⁻⁶. 9 CFU / mL ~ 4.0 × 10 9 The concentration of CFU / mL and the pH value are 3.5–4.0. The prepared seed solution is stored temporarily at 4°C and used within 24 hours.

[0064] Example 1:

[0065] This embodiment provides a method for preparing a fermented feed additive to improve the intestinal health of piglets, including the following steps:

[0066] (1) Solid precursor mixing: Weigh 50 kg of soybean meal, 30 kg of corn flour, and 20 kg of wheat bran, and grind them to the specified particle size. Weigh 5.0 kg of sodium alginate, 2.5 kg of chitosan, and 3.0 kg of β-tricalcium phosphate. Premix the sodium alginate, chitosan, and β-tricalcium phosphate evenly, and then put them into a twin-shaft paddle mixer to mix with soybean meal, corn flour, and wheat bran for 15 minutes until the mixing uniformity CV ≤ 5%.

[0067] (2) Inoculation and moisture adjustment: Measure 100L of sterile water (i.e., the material-to-water ratio is 1:1), and add the *Lactobacillus plantarum* seed solution (obtained in Preparation Example 1) to the sterile water at an inoculation rate of 3.0% (v / w, relative to the dry substrate mass) and mix well. Under stirring, spray the bacterial liquid into the solid mixture obtained in step (1) in the form of atomization. After spraying, adjust the initial pH of the system to 6.8 using a 1.0 mol / L NaOH solution.

[0068] (3) Metabolic triggering in-situ gelation fermentation: The conditioned material is placed in a fermentation bag with a one-way breathing valve and sealed, and then placed in a constant temperature fermentation chamber. The fermentation temperature is controlled at 35℃ and the fermentation time is 36 hours. No stirring is performed during the fermentation process, and in-situ gelation is triggered by metabolic acid production.

[0069] (4) Post-processing: After fermentation, the gel material is removed and placed in a freeze dryer. The pre-freezing temperature is -30℃ and the freezing time is 3 hours. The vacuum drying pressure is controlled at 15Pa and the desorption drying temperature is 25℃. The material is dried until the moisture content is 10%. The dried material is then pulverized and passed through a 20-mesh sieve to obtain the finished product.

[0070] Example 2:

[0071] This embodiment provides a method for preparing a fermented feed additive to improve the intestinal health of piglets, including the following steps:

[0072] (1) Solid precursor mixing: Weigh 60 kg of soybean meal, 25 kg of corn flour, and 15 kg of wheat bran, and grind them separately. Weigh 3.0 kg of sodium alginate, 4.0 kg of chitosan, and 2.0 kg of β-tricalcium phosphate. Mix all components evenly in a mixer at 60 rpm for 10 minutes.

[0073] (2) Inoculation and moisture adjustment: Measure 90L of sterile water (material-to-water ratio 1:0.9), add Lactobacillus plantarum seed solution, and the inoculation amount is 5.0% (v / w). Spray the bacterial solution into the solid material and adjust the initial pH to 6.5.

[0074] (3) Metabolic triggering in situ gelation fermentation: Seal the material and ferment it at 37°C for 24 hours.

[0075] (4) Post-processing: Same as in Example 1, using freeze drying process. After drying, the powder is pulverized and passed through a 40-mesh sieve.

[0076] Example 3:

[0077] This embodiment provides a method for preparing a fermented feed additive to improve the intestinal health of piglets, including the following steps:

[0078] (1) Solid precursor mixing: Weigh 40 kg of soybean meal, 30 kg of corn flour, and 30 kg of wheat bran, and grind them separately. Weigh 8.0 kg of sodium alginate, 1.0 kg of chitosan, and 5.0 kg of β-tricalcium phosphate. Mix all components evenly.

[0079] (2) Inoculation and moisture adjustment: Measure 110L of sterile water (material-to-water ratio 1:1.1), add Lactobacillus plantarum seed liquid, and the inoculation amount is 2.0% (v / w). Spray the bacterial solution into the solid material and adjust the initial pH to 7.0.

[0080] (3) Metabolic trigger in situ gelation fermentation: Seal the material and ferment it at 32°C for 40 hours.

[0081] (4) Post-processing: Same as in Example 1, using freeze drying process.

[0082] Example 4:

[0083] This embodiment provides a method for preparing a fermented feed additive to improve the intestinal health of piglets, including the following steps:

[0084] (1) Solid precursor mixing: Weigh 45 kg of soybean meal, 30 kg of corn flour, and 25 kg of wheat bran. Weigh 6.0 kg of sodium alginate, 3.0 kg of chitosan, and 4.0 kg of β-tricalcium phosphate. The mixing process is the same as in Example 1.

[0085] (2) Inoculation and moisture adjustment: The material-to-water ratio was set to 1:0.8, and the inoculation amount was 4.0% (v / w). The initial pH was adjusted to 6.6.

[0086] (3) Metabolic triggered in situ gelation fermentation: fermentation temperature 34℃, fermentation time 30 hours.

[0087] (4) Post-processing: Same as in Example 1.

[0088] Example 5:

[0089] This embodiment provides a method for preparing a fermented feed additive to improve the intestinal health of piglets, including the following steps:

[0090] (1) Solid precursor mixing: Weigh 50 kg of soybean meal, 25 kg of corn flour, and 25 kg of wheat bran. Weigh 5.0 kg of sodium alginate and 2.5 kg of chitosan. Use 3.0 kg of hydroxyapatite (HAP) instead of tricalcium β-phosphate. The rest of the operation is the same as in Example 1.

[0091] (2) Inoculation and moisture adjustment: The material-to-water ratio was 1:1.0, the inoculation amount was 3.0% (v / w), and the initial pH was 6.8.

[0092] (3) Metabolic triggered in situ gelation fermentation: fermentation temperature 35℃, fermentation time 36 hours.

[0093] (4) Post-processing: Same as in Example 1.

[0094] Example 6:

[0095] This embodiment provides a method for preparing a fermented feed additive to improve the intestinal health of piglets, including the following steps:

[0096] (1) Solid precursor mixing: The formula ratio is the same as in Example 1 (50% soybean meal, 30% corn flour, 20% wheat bran; 5 parts sodium alginate, 2.5 parts chitosan, and 3 parts β-tricalcium phosphate). Mix evenly.

[0097] (2) Inoculation and moisture adjustment: Measure 120L of sterile water (i.e., the material-to-water ratio is 1:1.2), add Lactobacillus plantarum seed liquid, and the inoculation amount is 3.0%. Adjust the initial pH to 6.8.

[0098] (3) Metabolic triggered in situ gelation fermentation: fermentation temperature 30℃, fermentation time 48 hours.

[0099] (4) Post-processing: After fermentation, the gel material is removed, cut into pieces, and placed in a vacuum drying oven. The vacuum degree is adjusted to -0.09MPa, the drying temperature is set to 45℃, and the drying time is 12 hours until the moisture content drops below 10%. Then, it is pulverized and passed through a 20-mesh sieve to obtain the final product.

[0100] Comparative Example 1:

[0101] Compared with Example 1, the difference is that sodium alginate, chitosan and β-tricalcium phosphate, three gelation precursors, are not added. That is, this formula is a conventional fermented feed. Everything else is the same.

[0102] Comparative Example 2:

[0103] Compared with Example 1, the differences are: (1) Sodium alginate, chitosan and β-tricalcium phosphate are not added to the solid precursor mixture. (2) After fermentation, the dried fermentation powder is mixed with equal amounts of sodium alginate, chitosan and β-tricalcium phosphate powder by simple mechanical and physical mixing without in-situ gelation, and the rest are the same.

[0104] Comparative Example 3:

[0105] Compared with Example 1, the difference is that β-tricalcium phosphate is replaced with calcium carbonate (CaCO3) of equal molar calcium, otherwise they are the same.

[0106] Comparative Example 4:

[0107] The difference from Example 1 is that chitosan is not added; otherwise, they are the same.

[0108] Comparative Example 5:

[0109] The difference from Example 1 is that β-tricalcium phosphate is not added; otherwise, they are the same.

[0110] Comparative Example 6:

[0111] The difference from Example 1 is that β-tricalcium phosphate is replaced with calcium lactate of equimolar calcium, otherwise they are the same.

[0112] Test Example 1:

[0113] The experimental method is as follows:

[0114] (1) Undried materials were obtained according to the preparation methods of Example 1, Comparative Example 3 and Comparative Example 6 respectively.

[0115] (2) Place approximately 1 kg of material into a fermenter equipped with an online pH electrode and an online rheometer (using a blade or spiral sensor). The fermenter has a jacketed temperature control function.

[0116] (3) The temperature of the fermentation tank is controlled at 35℃, and no mechanical stirring is performed during the fermentation process.

[0117] (4) Record the pH value, energy storage modulus (G') and loss modulus (G'') every 2 hours through the data acquisition system, and monitor continuously for 48 hours.

[0118] The test results are shown in Table 1 and Figure 1 As shown.

[0119] Table 1. Changes in pH and rheological parameters over time during fermentation:

[0120]

[0121] Table 1 shows that the fermentation processes of Example 1, Comparative Example 3, and Comparative Example 6 differ.

[0122] In Example 1, the pH of the system decreased from 6.82 to 5.12 during fermentation from 0 to 12 hours. During this stage, lactic acid bacteria produced acid. From 12 to 36 hours, the pH decrease slowed, remaining within the range of 4.08 to 4.58, forming a pH plateau. This pH plateau indicates that under acidic conditions, β-tricalcium phosphate dissolves, releasing calcium ions and phosphate buffer pairs to counteract lactic acid accumulation, thus stabilizing the pH of the fermentation system. Rheological data showed that the storage modulus G' of Example 1 increased after 12 hours of fermentation, rising from 35.7 Pa to 240.5 Pa after 48 hours, and the G' value remained higher than the loss modulus G''. A G' higher than G'' indicates that elastic behavior dominates, forming a gel network.

[0123] Comparative Example 3 used calcium carbonate. During its fermentation, the pH dropped to 5.30 from 0 to 12 hours, and then rose back to 6.30 from 18 to 24 hours. Calcium carbonate reacts under acidic conditions, releasing carbon dioxide and consuming acid, leading to a pH increase and inhibiting acid production by lactic acid bacteria. Rheological data showed that the G' value of Comparative Example 3 increased in the early stages, but after the pH recovered, the G' value failed to continue to increase, eventually remaining at a level of 34.0–45.2 Pa. The difference between G' and G'' was small, and sometimes G'' was higher than G', indicating that an effective gel network failed to form or that the structural strength was insufficient.

[0124] Comparative Example 6 used calcium lactate. During its fermentation, the pH value continuously decreased from 6.78 to 3.45 within 0 to 24 hours, reaching 3.25 at 48 hours. The curve did not show a pH buffer plateau. Calcium lactate, as a calcium source, did not provide a phosphate buffer pair, leading to a continuous decrease in the pH value of the fermentation system due to lactic acid accumulation, thus inhibiting lactic acid bacteria activity. Rheological data showed that the G' value of Comparative Example 6 increased slowly, eventually reaching 24.1–31.5 Pa. The close proximity of G' and G'' values, and sometimes G'' being higher than G', indicates that the gel network failed to form or had low structural strength under strongly acidic conditions.

[0125] In summary, Example 1 combines lactic acid bacteria fermentation and acid production with the gelation process through the buffering effect of β-tricalcium phosphate. This process achieves stable pH control and forms a gel network. Comparative Examples 3 and 6, due to pH fluctuations or lack of buffering capacity, had their gelation processes affected or failed to form effective structures.

[0126] Test Example 2:

[0127] The experimental method is as follows:

[0128] (1) Weigh 1g of the final product (dry powder) of all examples and comparative examples respectively.

[0129] (2) Prepare simulated gastric juice (SGF) with pH 2.0 according to the pharmacopoeia standard.

[0130] (3) Add each sample to a 50 mL Erlenmeyer flask containing SGF. Incubate at 37 °C and 150 rpm for 2 hours.

[0131] (4) After 2 hours of processing, remove the sample. Separate the sample into a solid phase (which may be a gel block or undissolved particles) and a liquid phase (SGF solution) by filtration or low-speed centrifugation (100g, 5 minutes).

[0132] (5) The solid and liquid phases were serially diluted, and the viable count of Lactobacillus plantarum (CFU / g or CFU / mL) was determined by MRS agar plate counting method.

[0133] (6) Weigh 1g of each sample dry powder, dissolve it in physiological saline, and immediately perform gradient dilution and plate counting to determine the initial total viable count at 0 hours.

[0134] (7) Calculate the viable cell survival rate according to the following formula: viable cell survival rate (%) = (number of viable cells in solid phase after 2 hours + number of viable cells in liquid phase after 2 hours) / (initial total number of viable cells at 0 hours) × 100%.

[0135] The test results are shown in Table 2.

[0136] Table 2. Survival rate of *Lactobacillus plantarum* after treatment in simulated gastric fluid (pH 2.0) for 2 hours:

[0137]

[0138] According to the data in Table 2, the viable survival rate of Lactobacillus plantarum varied among the samples after being treated in simulated gastric fluid (pH 2.0) for 2 hours.

[0139] Examples 1 through 6 all showed high viable cell survival rates, ranging from 76.2% to 85.9%. This indicates that the preparation method of the present invention constructs a sodium alginate-chitosan-calcium phosphate interpenetrating polymer network (IPN) gel structure through metabolism-triggered in-situ gelation, providing effective gastric acid protection for *Lactobacillus plantarum*. This IPN gel can physically encapsulate the bacterial cells, slowing down gastric acid penetration, and the calcium phosphate component in the gel dissolves in an acidic environment, releasing calcium ions and phosphates, further enhancing the acid stability of the gel network.

[0140] The viable bacterial survival rate of Comparative Example 1 (without gel precursor) was only 6.8%. This indicates that in the absence of a gel carrier, Lactobacillus plantarum is directly exposed to simulated gastric fluid, resulting in a large number of viable bacteria dying.

[0141] The viable cell survival rate of Comparative Example 2 (physically mixed gel precursor after fermentation) was 19.5%. Compared with Comparative Example 1, the survival rate was improved, indicating that the physically mixed gel precursor can provide partial protection. However, compared with the examples, its protective effect was significantly reduced, which verifies the importance of in-situ gelation for constructing a dense and uniform protective structure. Physical mixing failed to form a dense network structure of IPN gel, resulting in poor protective effect.

[0142] The viable bacterial survival rate in Comparative Example 3 (using calcium carbonate) was 25.1%. Combined with the results of Test Example 1, the violent reaction of calcium carbonate with gastric acid caused pH fluctuations, affecting the quality of gel formation. The unstable gel structure failed to effectively encapsulate and protect the bacterial cells.

[0143] The viable bacterial survival rate of Comparative Example 4 (without chitosan) was 48.7%. This survival rate was significantly lower than that of the Examples. This indicates that chitosan plays a role in the gel system, and the polyelectrolyte complex (PEC) network formed by chitosan and sodium alginate is key to constructing a complete IPN gel. The absence of this component leads to an incomplete gel structure and weakened protection against gastric acid.

[0144] The viable cell survival rate of Comparative Example 5 (without β-tricalcium phosphate) was 35.6%. This confirms the crucial role of β-tricalcium phosphate as a calcium ion source and pH buffer. The absence of β-tricalcium phosphate limits the ionic cross-linking of sodium alginate and lacks pH buffering during fermentation, affecting the formation and stability of the final gel network and thus reducing the protection of viable cells.

[0145] The viable cell survival rate of Comparative Example 6 (using calcium lactate) was 18.2%. Combined with the results of Test Example 1, calcium lactate did not provide effective pH buffering capacity, leading to a sharp drop in pH during fermentation. This strong acid pretreatment may have already damaged the cells, and the subsequent gel structure in simulated gastric fluid may not have provided sufficient protection, resulting in a low viable cell survival rate.

[0146] In summary, this invention utilizes a metabolically triggered in-situ gelation technique, leveraging the synergistic effects of sodium alginate, chitosan, and a pH-buffered ion-releasing agent (such as β-tricalcium phosphate), to construct an IPN gel network. This structure effectively resists the acidic environment of the stomach, significantly improving the stomach acid tolerance of *Lactobacillus plantarum*. Each key component (sodium alginate, chitosan, and β-tricalcium phosphate) and the in-situ gelation process contribute to enhancing the viability of the bacteria.

[0147] Test Example 3:

[0148] The experimental method is as follows:

[0149] (1) Take out each sample from the solid phase (gel block) that survived SGF treatment for 2 hours in Test Example 2.

[0150] (2) Thoroughly wash the solid phase of each sample with sterile physiological saline to remove residual SGF solution adhering to the surface.

[0151] (3) Prepare simulated intestinal fluid (SIF) at pH 6.8 according to standard methods.

[0152] (4) Transfer the cleaned solid phase of each sample to an Erlenmeyer flask containing 50 mL of SIF. Incubate in a shaking incubator at 37 °C and 100 rpm. Take 1 mL of sample from the SIF liquid at time points of 0.5 h, 1 h, 2 h, and 4 h.

[0153] (5) After serial dilution, the viable count of Lactobacillus plantarum in the SIF liquid phase (CFU / mL) was determined by MRS agar plate counting method. An equal amount of fresh SIF was added after sampling to maintain the total volume.

[0154] The test results are shown in Table 3.

[0155] Table 3 shows the concentration of *Lactobacillus plantarum* released after treatment with the product in simulated intestinal fluid (pH 6.8):

[0156]

[0157] According to the data in Table 3, the concentration of live bacteria released by each sample in simulated intestinal fluid (pH 6.8) was different.

[0158] Examples 1 through 6 all exhibited continuous release of live bacteria in simulated intestinal fluid. The concentration of live bacteria in the liquid phase increased over time, reaching a high level of 7.3 × 10⁻⁶ at 4 hours. 7 Up to 9.1×10 7 The concentration is between CFU / mL. This indicates that the preparation method of the present invention enables the product to achieve pH-responsive release in the intestinal environment. The formed IPN gel remains stable in the acidic environment of gastric juice, effectively protecting the bacterial cells, while in the higher pH environment of intestinal juice, the gel structure composed of sodium alginate and chitosan swells and / or moderately depolymerizes, thereby gradually releasing the embedded Lactobacillus plantarum. This release mechanism helps the probiotics function in the intestine.

[0159] The viable bacterial release concentration of Comparative Example 1 (without gel precursor) was only 0.4 × 10⁻⁶ after 4 hours. 5 CFU / mL. This is consistent with the results of Test Example 2. The lack of a gel carrier resulted in extremely low viable bacterial survival in the sample in SGF, thus limiting the number of cells available for release in SIF.

[0160] The viable cell release concentration of Comparative Example 2 (post-fermentation physically mixed gel precursor) reached 2.5 × 10⁻⁶ after 4 hours. 6 CFU / mL. Release levels were lower than in the example. Physical mixing failed to form an effective gel protective structure, resulting in a limited number of surviving cells after SGF treatment, which in turn affected the release levels in SIF.

[0161] Comparative Example 3 (using calcium carbonate) showed a viable bacterial release concentration of 1.5 × 10⁻⁶ at 4 hours. 6 The release rate was low (CFU / mL) because the drastic pH fluctuations caused by calcium carbonate affected the gel formation quality and bacterial survival, resulting in a reduced number of effectively released live bacteria.

[0162] The viable bacterial release concentration of Comparative Example 4 (without chitosan) was 4.8 × 10⁻⁶ after 4 hours. 7CFU / mL. The release rate was relatively low compared to the previous examples. This indicates the role of chitosan in IPN gel formation. The absence of chitosan may result in a less dense gel network structure, affecting both its protective ability against gastric acid and its release behavior in intestinal fluid.

[0163] The viable bacterial release concentration of Comparative Example 5 (without β-tricalcium phosphate) was 3.6 × 10⁻⁶ at 4 hours. 7 CFU / mL. This reflects the importance of β-tricalcium phosphate in the gel formation process. The absence of this component affects the ionic cross-linking of sodium alginate, leading to decreased gel structural stability, thereby affecting its protective effect in gastric juice and its effective release in intestinal juice.

[0164] Comparative Example 6 (using calcium lactate) showed a viable bacterial release concentration of only 1.0 × 10⁻⁶ at 4 hours. 6 CFU / mL. This result is consistent with the low survival rate observed in Test Example 2. Calcium lactate did not provide an effective pH buffer, resulting in an excessively low pH during fermentation, which damaged the cells and ultimately led to a small number of viable cells available for release.

[0165] In summary, the IPN gel prepared by the method of this invention can stably encapsulate live bacteria in acidic gastric juice and effectively release live bacteria in neutral intestinal juice. This pH-responsive release characteristic is its key advantage as a probiotic carrier.

[0166] Test Example 4:

[0167] The experimental method is as follows:

[0168] (1) Weigh 1g of the final product (dry powder) of all examples and comparative examples respectively.

[0169] (2) Prepare simulated gastric juice (SGF) with pH 2.0 according to the pharmacopoeia standard.

[0170] (3) Add each sample to a 50 mL Erlenmeyer flask containing SGF. Incubate at 37 °C and 150 rpm for 2 hours.

[0171] (4) After 2 hours of treatment, filter the sample through a 0.22 μm filter membrane and collect the filtrate (SGF solution).

[0172] (5) The concentration of lactic acid in the collected SGF liquid phase was determined by high performance liquid chromatography (HPLC).

[0173] The test results are shown in Table 4.

[0174] Table 4. Lactic acid release concentration of the product after 2 hours of treatment in simulated gastric fluid (pH 2.0):

[0175]

[0176] According to the data in Table 4, the lactic acid release concentrations of each sample differed after 2 hours of treatment in simulated gastric fluid (pH 2.0).

[0177] The lactic acid release concentrations in Examples 1 to 6 were low, ranging from 152 mg / L to 265 mg / L. This indicates that the IPN gel constructed in this invention can not only effectively encapsulate probiotics but also immobilize water-soluble metabolites such as lactic acid produced during fermentation within the gel network. This immobilization reduces the rapid release and degradation of lactic acid in gastric juice, achieving "bacteria-product co-protection," which helps lactic acid bacteria and their metabolites work synergistically. The density and stability of the gel network ensure effective blocking of small molecule metabolites in the acidic environment of the stomach.

[0178] The lactic acid release concentration in Comparative Example 1 (without added gel precursor) was 1870 mg / L. This indicates that in the absence of an effective encapsulation carrier, the lactic acid in the fermentation product was almost completely dissolved and released into the simulated gastric juice, with no fixation effect.

[0179] The lactic acid release concentration of Comparative Example 2 (post-fermentation physically mixed gel precursor) was 980 mg / L. This was lower than that of Comparative Example 1, but still much higher than that of the Example. This indicates that although the physically mixed gel precursor can provide some adsorption or inhibition, it failed to form a stable IPN gel network and could not effectively prevent the penetration and release of lactic acid.

[0180] The lactic acid release concentration in Comparative Example 3 (using calcium carbonate) was 1150 mg / L. This is related to the instability of the gel structure caused by calcium carbonate in Test Example 1. Unstable gel networks have high porosity and weak ability to encapsulate lactic acid.

[0181] Comparative Example 4 (without chitosan) showed a lactic acid release concentration of 620 mg / L. This value is higher than that of the Example. This indicates that in the absence of the polyelectrolyte complex (PEC) network formed by chitosan, the gel's density decreases, its porosity increases, and its ability to encapsulate lactic acid is weakened.

[0182] Comparative Example 5 (without β-tricalcium phosphate) showed a lactic acid release concentration of 750 mg / L. This reflects the crucial role of β-tricalcium phosphate in IPN gel formation. The lack of β-tricalcium phosphate affected the ionic cross-linking of sodium alginate, resulting in insufficient gel structure strength and integrity, thereby reducing the fixation effect on lactic acid.

[0183] Comparative Example 6 (using calcium lactate) showed a lactic acid release concentration of 1380 mg / L. Combined with Test Example 1, calcium lactate did not provide a pH buffer, resulting in an excessively low fermentation pH, which may have affected gel network formation and consequently reduced the ability to encapsulate lactic acid.

[0184] In summary, the metabolism-triggered in-situ gelation technology of this invention, by constructing a stable IPN gel network, can effectively immobilize water-soluble metabolites such as lactic acid produced during fermentation, significantly reducing their release in simulated gastric juice. This result further demonstrates the advantages of this invention in achieving synergistic protection of probiotics and their metabolites.

[0185] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A fermented feed additive for improving the intestinal health of piglets, characterized in that, The additive is a gel-state fermentation product with an interpenetrating polymer network structure, made from raw materials comprising the following parts by weight: 95–105 parts of solid-state fermentation substrate; 3-8 parts of sodium alginate; 1-4 parts chitosan; 2-5 parts of β-tricalcium phosphate; 2-5 parts of Lactobacillus plantarum seed culture; The viable cell concentration of the *Lactobacillus plantarum* seed solution was 2.0 × 10⁻⁶. 9 CFU / mL ~ 4.0 × 10 9 CFU / mL; In this process, sodium alginate, chitosan, and β-tricalcium phosphate serve as gel precursors. During fermentation, β-tricalcium phosphate releases calcium and phosphate ions through acid production via Lactobacillus plantarum metabolism, and then cross-links in situ to form an interpenetrating polymer network gel structure containing Lactobacillus plantarum and its metabolites.

2. The fermented feed additive for improving the intestinal health of piglets according to claim 1, characterized in that, The raw material consists of the following components in parts by weight: 45-60 parts soybean meal, 25-30 parts corn flour, and 15-30 parts wheat bran; 5-6 parts sodium alginate; Chitosan 2.5-3 parts; 3-4 parts of β-tricalcium phosphate; 3-4 parts of Lactobacillus plantarum seed culture.

3. The fermented feed additive for improving the intestinal health of piglets according to claim 1, characterized in that, The sodium alginate has a mannuronic acid / guluronic acid ratio of 0.5 to 1.0, and its 1% aqueous solution has an apparent viscosity of 100 to 300 mPa·s at 20°C; the chitosan has a degree of deacetylation ≥85% and a weight-average molecular weight of 50 to 150 kDa.

4. The fermented feed additive for improving the intestinal health of piglets according to claim 1, characterized in that, The viable bacterial survival rate of the fermented feed additive is ≥75% after being treated in simulated gastric juice at pH 2.0 for 2 hours, and the lactic acid release concentration in the simulated gastric juice after treatment is ≤300mg / L.

5. The fermented feed additive for improving the intestinal health of piglets according to claim 1, characterized in that, The β-tricalcium phosphate begins to dissolve and release calcium ions and phosphate buffer pairs when the pH of the fermentation system drops below 5.5, maintaining the pH value between 4.0 and 4.6 in the later stage of fermentation.

6. A method for preparing a fermented feed additive for improving intestinal health in piglets as described in any one of claims 1-5, characterized in that, Includes the following steps: S1. Solid precursor mixing: Sodium alginate, chitosan and β-tricalcium phosphate are premixed evenly and then mixed with the pulverized solid fermentation substrate to obtain solid mixture. S2. Inoculation and conditioning: Disperse the Lactobacillus plantarum seed liquid in sterile water, add it to the solid mixture in the form of a spray, and adjust the initial pH to 6.5-7.0; S3, Metabolic Triggered In-Situ Gel Fermentation: The material obtained in S2 is sealed and allowed to ferment statically at 30-37℃ for 24-48 hours. No mechanical stirring is performed during the fermentation process. In-situ gelation is initiated by metabolic acid production. S4. Post-processing: After fermentation, the gel-like material is dried and pulverized to obtain the finished product.

7. The preparation method according to claim 6, characterized in that, The solid fermentation substrate includes soybean meal, corn flour and wheat bran, and the mixing uniformity CV after mixing is ≤5%; in step S2, the amount of sterile water added is used to control the material-to-water ratio between 1:0.8 and 1:1.

2.

8. The preparation method according to claim 6, characterized in that, In step S2, the initial pH is adjusted using NaOH solution to ensure that the initial environment is suitable for the growth of Lactobacillus plantarum and does not trigger the acidic gelation of sodium alginate; in step S3, the pH of the system is controlled between 4.0 and 4.2 at the end of fermentation.

9. The preparation method according to claim 6, characterized in that, In step S4, the drying process adopts a vacuum freeze-drying process with the following specific parameters: pre-freezing temperature below -30℃, vacuum drying pressure ≤15Pa, desorption drying temperature ≤25℃, and drying to a moisture content ≤10%.

10. The preparation method according to claim 6, characterized in that, In step S3 of the preparation method, the release of calcium ions and pH buffering are achieved simultaneously through the dissolution of β-tricalcium phosphate, so that the storage modulus of the fermentation system reaches more than 150 Pa at the end of fermentation, and the storage modulus is greater than the loss modulus.

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