Composite probiotic agent and application thereof in improving flavor of corn puree

Abstract

The invention relates to the field of bacterial strains, and discloses a composite probiotic agent and application thereof in improving the flavor of corn puree. According to the composite probiotic agent, sodium alginate and chitosan are subjected to functional group modification to form an embedding material with enhanced hydrophobicity and water solubility, and a pH-sensitive double-layer microcapsule structure is constructed by synergistically embedding plant lactobacillus, lactobacillus mucus fermentation and saccharomyces cerevisiae. The probiotic agent is applied to fermentation of the corn puree, probiotics can be effectively protected to pass through a gastric acid environment, targeted release of intestinal tracts is achieved, meanwhile, the corn puree is promoted to generate rich flavor substances through multi-bacterium synergistic fermentation, and the sweet feeling, milk fragrance and the overall flavor level of a product are remarkably improved. The process conditions are mild, large-scale production is easy, and the obtained corn puree beverage is rich and mellow in flavor and has the probiotic function.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a compound probiotic agent and its application in improving the flavor of corn pulp. Background Technology

[0002] With the popularization of healthy eating concepts, probiotics have received much attention for their effects such as regulating gut microbiota and enhancing immunity. Corn pulp, as a natural grain beverage, is rich in dietary fiber, vitamins, and minerals and has high nutritional value. However, its natural flavor is monotonous, its taste is rough, and problems such as starch retrogradation and fishy smell are easily generated during processing, which limits its market acceptance.

[0003] Currently, there are some products or methods on the market that improve the flavor of corn pulp by adding probiotics. For example, fermentation is carried out using a single lactic acid bacteria or yeast strain, or commercial probiotic powder is added to the corn pulp. However, these methods have significant shortcomings: first, ordinary probiotics have a low survival rate in the acidic environment of corn pulp, making it difficult to exert a sustained fermentation effect; second, the flavor substances produced by single-strain fermentation are limited, resulting in a negligible improvement in taste; and third, unencapsulated probiotics are easily inactivated in gastric acid, failing to effectively reach the intestines to exert their beneficial functions. Furthermore, while the traditional sodium alginate-chitosan encapsulation system provides some protection, the sodium alginate gel does not shrink sufficiently in gastric acid, and chitosan has poor water solubility, leading to low encapsulation efficiency and weak targeting.

[0004] To address the aforementioned problems, this invention modifies the functional groups of the encapsulation wall material and optimizes the combination of probiotics, designing a compound probiotic agent with high survival rate and strong targeting. When applied to corn pulp fermentation, this probiotic agent significantly enhances the flavor profile and smoothness of the product, while ensuring effective colonization of probiotics in the gut. Summary of the Invention

[0005] The purpose of this invention is to provide a compound probiotic agent and its application in improving the flavor of corn pulp. This compound probiotic agent uses modified sodium alginate and modified chitosan to synergistically encapsulate *Lactobacillus plantarum*, *Lactobacillus fermentum*, and *Saccharomyces cerevisiae*, forming a pH-sensitive double-layer microcapsule structure. This not only significantly improves the survival rate of probiotics in gastric acid and achieves targeted release into the intestines, but also promotes the generation of rich flavor substances in the corn pulp through multi-strain synergistic fermentation, thereby obtaining a corn pulp beverage with a mellow taste and rich flavor.

[0006] In a first aspect, the present invention provides a compound probiotic agent comprising compound probiotics encapsulated by modified sodium alginate and modified chitosan, wherein the compound probiotics include Lactobacillus plantarum, Lactobacillus fermentum, and Saccharomyces cerevisiae.

[0007] Preferably, the probiotic agent has a pH-sensitive bilayer microcapsule structure, including a core and a shell; the core is formed by encapsulating probiotics with modified sodium alginate, and the shell is formed by coating the surface of the core with modified chitosan.

[0008] Preferably, the modified sodium alginate is sodium alginate modified with octenyl succinic anhydride.

[0009] Preferably, the modified chitosan is carboxymethyl chitosan.

[0010] Preferably, the volume ratio of *Lactobacillus plantarum*, *Lactobacillus fermentum*, and *Saccharomyces cerevisiae* is 1:(0.5-2):(0.5-2). More preferably, the volume ratio of *Lactobacillus plantarum*, *Lactobacillus fermentum*, and *Saccharomyces cerevisiae* is 1:1:1; even more preferably, the bacterial concentrations of *Lactobacillus plantarum*, *Lactobacillus fermentum*, and *Saccharomyces cerevisiae* are the same, and the bacterial concentration is 1×10⁻⁶. 9 -1×10 10 CFU / mL. *Lactobacillus plantarum* CICC 20265, *Lactobacillus fermentum* CICC 21800, and *Saccharomyces cerevisiae* CICC 1374 were used.

[0011] Secondly, this invention provides a method for preparing a compound probiotic agent and a method for applying the compound probiotic agent in corn pulp. The specific preparation scheme is as follows: S1. Preparation of modified sodium alginate: Weigh 10-30 g of sodium alginate powder and dissolve it in 200-500 ml of deionized water. Place the solution in a water bath at 50-70℃ and stir at 200-400 r / min until completely dissolved. Add 5-15 mL of octenyl succinic anhydride. Monitor the pH of the reaction solution with a pH meter and adjust the pH to 8.0-9.0 with 1 mol / L NaOH solution. During the reaction, maintain the pH of the system within the range of 8.0-9.0 by adding NaOH solution dropwise. Continue stirring for 2-4 hours. After the reaction is complete, pour the reaction solution into 2-3 times the volume of 95% ethanol to precipitate the solid. Filter the precipitate using a Buchner funnel. Wash the obtained solid 2-3 times with 70% ethanol, then transfer it to a vacuum drying oven and dry it at 40-60℃ for 4-8 hours to obtain hydrophobically modified sodium alginate powder. This step introduces octenyl succinic acid groups into the sodium alginate molecular chain through esterification, enhancing the hydrophobicity and acid resistance of the material and providing a more stable wall material for subsequent embedding.

[0012] S2. Preparation of Modified Chitosan: Weigh 5-15 g of chitosan powder and add it to 100-300 mL of isopropanol. Stir and disperse at 300-500 r / min for 30 minutes. Add 5-10 mL of 10 M sodium hydroxide solution and alkalize in an ice-water bath for 1 hour. Then add 10-30 g of sodium chloroacetate and place the mixture in a 50-70℃ water bath, stirring continuously for 3-6 hours. After the reaction is complete, pour the reaction solution into 2-3 times its volume of 95% ethanol to precipitate, filter, wash the solid 2-3 times with 70% ethanol, and then place it in a vacuum drying oven and dry at 40-60℃ for 4-8 hours to obtain carboxymethyl chitosan powder. This step introduces carboxymethyl groups into the chitosan molecule through carboxymethylation, significantly improving its water solubility and bioadhesion, and enhancing the retention and release performance of microcapsules in the intestine.

[0013] S3. Preparation of the compound probiotic suspension: *Lactobacillus plantarum* and *Lactobacillus fermentum* were inoculated into MRS liquid medium, and *Saccharomyces cerevisiae* was inoculated into YPD liquid medium. All were incubated statically at 35-40℃ for 18-24 hours. Each suspension was centrifuged at 4000-6000 r / min for 8-12 minutes, the supernatant was discarded, and the bacterial cells were resuspended in sterile physiological saline. The bacterial concentration was adjusted to 1×10⁻⁶ using a hemocytometer. 9 -1×10 10 CFU / mL. The three bacterial suspensions were mixed at a volume ratio of 1:1:1 to obtain a compound probiotic suspension. This step yields a highly active and balanced compound probiotic, providing core live bacteria material for encapsulation.

[0014] S4. Encapsulation of the compound probiotic agent: Weigh 2-5 g of the modified sodium alginate powder obtained in step S1, dissolve it in 100-200 mL of deionized water, and stir in a water bath at 40-60℃ until completely dissolved. Add 10-30 mL of the compound probiotic suspension obtained in step S3, and mix evenly with a magnetic stirrer at 300-500 r / min. Pump the above mixture into the container at a flow rate of 0.5-1.0 mL / h using an electrostatic droplet generator, forming uniform microdroplets under a voltage of 8-12 kV. Drop these microdroplets into a container containing 2-4% CaCl2 solution (placed on a magnetic stirrer and slowly stirred at 100-150 r / min). After contact with Ca²⁺, the droplets rapidly gel into spheres and crosslink for 10-20 minutes. Filter out the microcapsules with a sieve and rinse twice with deionized water. The microcapsules were then immersed in a 0.5-1.5% carboxymethyl chitosan solution obtained in step S2, and the coating was gently stirred for 5-15 minutes. After removal, the microcapsules were cross-linked with a 2% CaCl2 solution for 5 minutes, filtered, and the resulting encapsulated product was placed in a freeze dryer and dried at -40℃ to -50℃ and a vacuum of 10-20 Pa for 24-48 hours to obtain the composite probiotic agent. The undried, wet microcapsule suspension after cross-linking and curing with CaCl2 solution was appropriately diluted and measured using a laser particle size analyzer. This step involves preparing uniformly sized microdroplets using an electrostatic droplet generation method, followed by calcium ion cross-linking and layer-by-layer coating to construct a pH-responsive bilayer encapsulation structure, achieving protection of probiotics in the gastric acid environment and targeted release into the intestinal tract.

[0015] S5. Application of Compound Probiotic Agent in Corn Slurry: Select fresh or dried corn kernels. If using dried corn kernels, pre-soak them until fully swelled and absorb water. After washing the raw materials, add them to a pulping machine and pulp to obtain corn slurry. Detect the solids content of the corn slurry, dilute or concentrate with water to adjust the solids content to 10-15%. Place the adjusted corn slurry in a jacketed kettle, heat to 90-100℃, maintain for 10-20 minutes for sterilization, and then cool to 35-40℃. Add the compound probiotic agent obtained in step S4 at 0.1-0.5% of the total mass of the corn slurry, stir evenly, and ferment in a constant temperature fermentation tank at 35-40℃ for 12-24 hours. After fermentation, refrigerate the product at 4℃ to obtain a flavor-improved corn slurry beverage. This step utilizes the synergistic fermentation effect of compound probiotics to convert starch, protein, and other substances in corn pulp into small-molecule sugars, amino acids, and flavor compounds, significantly enhancing the sweetness, milkiness, and overall flavor profile of the beverage.

[0016] Preferably, in step S1, the amount of sodium alginate is 15 g, the volume of deionized water is 300 mL, the reaction temperature is 60 °C, the amount of octenyl succinic anhydride added is 10 mL, and the reaction time is 3 hours.

[0017] Preferably, in step S2, the amount of chitosan used is 10 g, the volume of isopropanol is 200 mL, the amount of sodium chloroacetate added is 20 g, the reaction temperature is 60℃, and the reaction time is 4.5 hours.

[0018] Preferably, in step S3, the concentrations of *Lactobacillus plantarum*, *Lactobacillus fermentum*, and *Saccharomyces cerevisiae* are all 5 × 10⁻⁶. 9 CFU / mL, with a mixing volume ratio of 1:1:1.

[0019] Preferably, in step S4, the amount of modified sodium alginate is 3.5 g, the volume of deionized water is 150 mL, the amount of compound probiotic suspension added is 20 mL, the concentration of CaCl2 solution is 3%, and the cross-linking time is 15 minutes.

[0020] Preferably, in step S4, the concentration of the carboxymethyl chitosan solution is 1%, and the coating time is 10 minutes.

[0021] Preferably, in step S5, the raw material for preparing corn pulp is fresh sweet corn kernels or soaked dried corn kernels, the solid content of the adjusted corn pulp is 12%, the sterilization conditions are heating at 95°C for 15 minutes, the inoculation amount of compound probiotic agent is 0.3%, the fermentation temperature is 37°C, and the fermentation time is 18 hours.

[0022] Preferably, in step S5, after fermentation, the corn pulp beverage is refrigerated at 4°C.

[0023] Advantages of this invention: 1. This invention significantly improves the acid resistance, water solubility and bioadhesion of the encapsulation material by modifying the functional groups of sodium alginate and chitosan, forming a stable pH-sensitive bilayer microcapsule that effectively protects probiotics through the acidic environment of the stomach and enables precise release into the intestine.

[0024] 2. This invention uses a compound strain of Lactobacillus plantarum, Lactobacillus fermentum and Saccharomyces cerevisiae. Through multi-strain synergistic fermentation, corn pulp is promoted to generate rich flavor substances such as lactic acid, acetic acid and acetoin, which greatly improves the sweetness, milky aroma and overall flavor of the product.

[0025] 3. The preparation process of the compound probiotic agent of the present invention is mild, the steps are clear, the instruments used are conventional, it is suitable for large-scale production, and the product has a high survival rate of live bacteria and good storage stability.

[0026] 4. This invention applies compound probiotic agents to the fermentation of corn pulp, which not only enhances the flavor and taste of the beverage, but also endows the product with probiotic functions, achieving a dual upgrade in nutrition and flavor, and meeting the needs of modern consumers for healthy and delicious drinks. Attached Figure Description

[0027] Figure 1 Bar chart showing the survival rates of *Lactobacillus plantarum*, *Lactobacillus fermentum*, and *Saccharomyces cerevisiae* after different samples were treated in simulated gastric fluid.

[0028] Figure 2 Line graph showing the cumulative release rate of live bacteria in simulated intestinal fluid for different samples.

[0029] Figure 3 Bar chart showing the colonization effect of different probiotic agents in the mouse intestine on days 7 and 14.

[0030] Figure 4 Comparison of the content of major flavor substances in corn pulp under different fermentation conditions.

[0031] Figure 5 Radar chart comparing sensory evaluation scores of different fermentation samples.

[0032] Figure 6 : Particle size distribution diagram of compound probiotic microcapsules.

[0033] Figure 7 , Figure 8 Photo of corn pulp without adjusted solids content. Detailed Implementation

[0034] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features described therein can be combined with each other.

[0035] The sources and specifications of the raw materials used in the embodiments of this application are as follows: Sodium alginate was purchased from Qingdao Mingyue Seaweed Group Co., Ltd., and the product is food grade. Chitosan was purchased from Zhejiang Jinke Biochemical Co., Ltd., with a degree of deacetylation ≥90%. Octenyl succinic anhydride was purchased from Hangzhou Zhongxiang Chemical Co., Ltd., food grade; Sodium chloroacetate was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. The embodiments of this application use *Lactobacillus plantarum* CICC 20265, *Lactobacillus fermentum* CICC 21800, and *Saccharomyces cerevisiae* CICC 1374, all of which were purchased from the China Industrial Microbial Culture Collection Center. MRS and YPD media were purchased from Beijing Aoboxing Biotechnology Co., Ltd.

[0036] Example 1 S1. Preparation of modified sodium alginate: Weigh 15 g of sodium alginate powder and dissolve it in 300 mL of deionized water. Place the solution in a 60℃ water bath and stir at 300 r / min until completely dissolved. Add 10 mL of octenyl succinic anhydride. Monitor the pH of the reaction solution with a pH meter and adjust the pH to 8.5 with 1 mol / L NaOH solution. Maintain the pH of the system at 8.5 by adding NaOH solution dropwise during the reaction. Continue stirring for 3 hours. After the reaction is complete, pour the reaction solution into 2.5 times its volume of 95% ethanol to precipitate the solid. Filter the solid using a Buchner funnel. Wash the solid twice with 70% ethanol and then transfer it to a vacuum drying oven. Dry the solid at 50℃ for 6 hours to obtain hydrophobically modified sodium alginate powder. This step introduces octenyl succinic acid groups into the sodium alginate molecular chain through esterification, enhancing the hydrophobicity and acid resistance of the material and providing a more stable wall material for subsequent embedding.

[0037] S2. Preparation of modified chitosan: Weigh 10 g of chitosan powder and add it to 200 mL of isopropanol. Stir and disperse at 400 r / min for 30 minutes. Add 6 mL of 10 M sodium hydroxide solution and alkalize in an ice-water bath for 1 hour. Then add 20 g of sodium chloroacetate and place the mixture in a 60℃ water bath, stirring continuously for 4.5 hours. After the reaction is complete, pour the reaction solution into 2.5 times its volume of 95% ethanol to precipitate, filter, wash the solid twice with 70% ethanol, and then place it in a vacuum drying oven and dry at 50℃ for 6 hours to obtain carboxymethyl chitosan powder. This step introduces carboxymethyl groups into the chitosan molecule through carboxymethylation, which significantly improves its water solubility and bioadhesion, and enhances the retention and release performance of microcapsules in the intestine.

[0038] S3. Preparation of the compound probiotic suspension: *Lactobacillus plantarum* and *Lactobacillus fermentum* were inoculated into MRS liquid medium, and *Saccharomyces cerevisiae* was inoculated into YPD liquid medium. All were incubated statically at 37℃ for 21 hours. Each bacterial suspension was centrifuged at 5000 r / min for 10 minutes, the supernatant was discarded, and the bacterial cells were resuspended in sterile physiological saline. The bacterial concentration was adjusted to 5 × 10⁻⁶ using a hemocytometer. 9 CFU / mL. The three bacterial suspensions were mixed at a volume ratio of 1:1:1 to obtain a compound probiotic suspension. This step yields a highly active and balanced compound probiotic, providing core live bacteria material for encapsulation.

[0039] S4. Encapsulation of the compound probiotic agent: Weigh 3.5 g of the modified sodium alginate powder obtained in step S1, dissolve it in 150 mL of deionized water, and stir in a 50°C water bath until completely dissolved. Add 20 mL of the compound probiotic suspension obtained in step S3, and mix thoroughly with a magnetic stirrer at 400 r / min. Pump the above mixture into the container at a flow rate of 0.6 mL / h using an electrostatic droplet generator, forming uniform microdroplets under a 9 kV voltage. Drop these microdroplets into a container containing 2.5% CaCl2 solution (placed on a magnetic stirrer and slowly stirred at 110 r / min). The droplets rapidly gel into spheres upon contact with Ca²⁺, and crosslink for 15 minutes. Filter the microcapsules through a sieve and rinse twice with deionized water. The microcapsules were then immersed in a 1.0% carboxymethyl chitosan solution obtained in step S2, and the coating was gently stirred for 10 minutes. After removal, the coating was cross-linked with a 2% CaCl2 solution for 5 minutes, filtered, and the resulting encapsulated product was placed in a freeze dryer and dried at -45℃ and a vacuum of 15 Pa for 36 hours to obtain the composite probiotic agent. The wet microcapsule suspension after cross-linking and curing with CaCl2 solution, which was not dried, was appropriately diluted and measured using a laser particle size analyzer. The particle size determination results of the wet microcapsules prepared in this step are as follows: Figure 6 As shown in the diagram, this step involves preparing uniformly sized microdroplets using an electrostatic droplet generation method, followed by calcium ion cross-linking and layer-by-layer coating to construct a pH-responsive bilayer encapsulation structure. This achieves the protection of probiotics in the acidic gastric environment and their targeted release into the intestinal tract. S5. Application of compound probiotics in corn pulp: Select fresh corn kernels, wash and pulp them (e.g., ...). Figure 7 , Figure 8 As shown in the figure, adjust the solids content to 12%; place the corn pulp with a solids content of 12% in a jacketed kettle, heat to 95℃, maintain for 15 minutes for sterilization, and then cool to 37℃. Add the compound probiotic agent obtained in step S4 at 0.3% of the total mass of the corn pulp with a solids content of 12%, stir well, and ferment in a constant temperature fermentation tank at 37℃ for 18 hours. After fermentation, refrigerate the product at 4℃ to obtain a corn pulp beverage with improved flavor. This step utilizes the synergistic fermentation effect of the compound probiotic agent to convert starch, protein, etc. in the corn pulp into small molecule sugars, amino acids, and flavor compounds, significantly improving the sweetness, milky aroma, and overall flavor profile of the beverage.

[0040] Example 2 S1. Preparation of modified sodium alginate: Weigh 10 g of sodium alginate powder and dissolve it in 200 mL of deionized water. Place the solution in a 50℃ water bath and stir at 200 r / min until completely dissolved. Add 5 mL of octenyl succinic anhydride. Monitor the pH of the reaction solution with a pH meter and adjust the pH to 8.0 with 1 mol / L NaOH solution. Maintain the pH of the system at 8.0 by adding NaOH solution dropwise during the reaction. Continue stirring for 2 hours. After the reaction is complete, pour the reaction solution into twice the volume of 95% ethanol to precipitate the solid. Filter the solid using a Buchner funnel. Wash the solid twice with 70% ethanol and then transfer it to a vacuum drying oven. Dry the solid at 40℃ for 4 hours to obtain hydrophobically modified sodium alginate powder. This step introduces octenyl succinic acid groups into the sodium alginate molecular chain through esterification, enhancing the hydrophobicity and acid resistance of the material and providing a more stable wall material for subsequent embedding.

[0041] S2. Preparation of modified chitosan: Weigh 5 g of chitosan powder and add it to 100 mL of isopropanol. Stir and disperse at 300 r / min for 30 minutes. Add 5 mL of 10 M sodium hydroxide solution and alkalize in an ice-water bath for 1 hour. Then add 10 g of sodium chloroacetate and place the mixture in a 50℃ water bath, stirring continuously for 3 hours. After the reaction is complete, pour the reaction solution into twice the volume of 95% ethanol to precipitate, filter, wash the solid twice with 70% ethanol, and then place it in a vacuum drying oven and dry at 40℃ for 4 hours to obtain carboxymethyl chitosan powder. This step introduces carboxymethyl groups into the chitosan molecule through carboxymethylation, which significantly improves its water solubility and bioadhesion, and enhances the retention and release performance of microcapsules in the intestine.

[0042] S3. Preparation of the compound probiotic suspension: *Lactobacillus plantarum* and *Lactobacillus fermentum* were inoculated into MRS liquid medium, and *Saccharomyces cerevisiae* was inoculated into YPD liquid medium. All were incubated statically at 35℃ for 18 hours. Each bacterial suspension was centrifuged at 4000 r / min for 8 minutes, the supernatant was discarded, and the bacterial cells were resuspended in sterile physiological saline. The bacterial concentration was adjusted to 1×10⁻⁶ using a hemocytometer. 9 CFU / ml. The three bacterial suspensions were mixed at a volume ratio of 1:1:1 to obtain a compound probiotic suspension. This step yields a highly active and balanced compound probiotic, providing core live bacteria material for encapsulation.

[0043] S4. Encapsulation of the compound probiotic agent: Weigh 2 g of the modified sodium alginate powder obtained in step S1, dissolve it in 100 mL of deionized water, and stir in a 40℃ water bath until completely dissolved. Add 10 mL of the compound probiotic suspension obtained in step S3, and mix evenly with a magnetic stirrer at 300 r / min. Pump the above mixture into the container at a flow rate of 0.5 mL / h using an electrostatic droplet generator, forming uniform microdroplets under 8 kV voltage. Drop these microdroplets into a container containing 2% CaCl2 solution (placed on a magnetic stirrer and slowly stirred at 100 r / min). After contact with Ca²⁺, the droplets rapidly gel into spheres and crosslink for 10 minutes. Filter the microcapsules through a sieve and rinse twice with deionized water. The microcapsules were then immersed in a 0.5% carboxymethyl chitosan solution obtained in step S2, and the coating was gently stirred for 5 minutes. After removal, the microcapsules were cross-linked with a 2% CaCl2 solution for 5 minutes, filtered, and the resulting encapsulated product was placed in a freeze dryer and dried at -40℃ and a vacuum of 10 Pa for 24 hours to obtain the composite probiotic agent. The wet microcapsule suspension after cross-linking and curing with CaCl2 solution, which was not dried, was appropriately diluted and measured using a laser particle size analyzer. This step involves preparing uniformly sized microdroplets using an electrostatic droplet generation method, followed by calcium ion cross-linking and layer-by-layer coating to construct a pH-responsive bilayer encapsulation structure, achieving protection of probiotics in the gastric acid environment and targeted release into the intestinal tract.

[0044] S5. Application of Compound Probiotic Agent in Corn Pulp: Select fresh corn kernels, wash and pulp them, adjusting the solids content to 12%. Place the corn pulp with a 12% solids content in a jacketed kettle, heat to 90℃, maintain for 10 minutes for sterilization, and then cool to 35℃. Add the compound probiotic agent obtained in step S4 at 0.1% of the total mass of the corn pulp with a 12% solids content, stir evenly, and ferment in a 35℃ constant temperature fermentation tank for 12 hours. After fermentation, refrigerate the product at 4℃ to obtain a flavor-improved corn pulp beverage. This step utilizes the synergistic fermentation effect of the compound probiotic agent to convert starch and protein in the corn pulp into small molecule sugars, amino acids, and flavor compounds, significantly enhancing the sweetness, milky aroma, and overall flavor profile of the beverage.

[0045] Example 3 S1. Preparation of Modified Sodium Alginate: Weigh 20 g of sodium alginate powder and dissolve it in 400 mL of deionized water. Place the solution in a 65℃ water bath and stir at 350 r / min until completely dissolved. Add 12 mL of octenyl succinic anhydride. Monitor the pH of the reaction solution with a pH meter and adjust the pH to 8.7 with 1 mol / L NaOH solution. Maintain the pH of the system at 8.7 by adding NaOH solution dropwise during the reaction. Continue stirring for 3.5 hours. After the reaction is complete, pour the reaction solution into 2.7 times its volume of 95% ethanol to precipitate the solid. Filter the precipitate using a Buchner funnel. Wash the obtained solid three times with 70% ethanol and then transfer it to a vacuum drying oven. Dry the solid at 55℃ for 7 hours to obtain hydrophobically modified sodium alginate powder. This step introduces octenyl succinic acid groups into the sodium alginate molecular chain through esterification, enhancing the hydrophobicity and acid resistance of the material and providing a more stable wall material for subsequent embedding.

[0046] S2. Preparation of modified chitosan: Weigh 12 g of chitosan powder and add it to 250 mL of isopropanol. Stir and disperse at 450 r / min for 30 minutes. Add 8 mL of 10 M sodium hydroxide solution and alkalize in an ice-water bath for 1 hour. Then add 25 g of sodium chloroacetate and place the mixture in a 65℃ water bath, stirring continuously for 5 hours. After the reaction is complete, pour the reaction solution into 2.7 times its volume of 95% ethanol to precipitate, filter, wash the solid three times with 70% ethanol, and then place it in a vacuum drying oven and dry at 55℃ for 7 hours to obtain carboxymethyl chitosan powder. This step introduces carboxymethyl groups into the chitosan molecule through carboxymethylation, which significantly improves its water solubility and bioadhesion, and enhances the retention and release performance of microcapsules in the intestine.

[0047] S3. Preparation of the compound probiotic suspension: *Lactobacillus plantarum* and *Lactobacillus fermentum* were inoculated into MRS liquid medium, and *Saccharomyces cerevisiae* was inoculated into YPD liquid medium. All were incubated statically at 38℃ for 22 hours. Each bacterial suspension was centrifuged at 5500 r / min for 11 minutes, the supernatant was discarded, and the bacterial cells were resuspended in sterile physiological saline. The bacterial concentration was adjusted to 8 × 10⁻⁶ using a hemocytometer. 9 CFU / mL. The three bacterial suspensions were mixed at a volume ratio of 1:1:1 to obtain a compound probiotic suspension. This step yields a highly active and balanced compound probiotic, providing core live bacteria material for encapsulation.

[0048] S4. Encapsulation of the compound probiotic agent: Weigh 4 g of the modified sodium alginate powder obtained in step S1, dissolve it in 180 mL of deionized water, and stir in a 55℃ water bath until completely dissolved. Add 25 mL of the compound probiotic suspension obtained in step S3, and mix evenly with a magnetic stirrer at 450 r / min. Pump the above mixture into the container at a flow rate of 0.8 mL / h using an electrostatic droplet generator, forming uniform microdroplets under a 10 kV voltage. Drop these microdroplets into a container containing 3.5% CaCl2 solution (placed on a magnetic stirrer and slowly stirred at 140 r / min). The droplets rapidly gel into spheres upon contact with Ca²⁺, and crosslink for 18 minutes. Filter the microcapsules through a sieve and rinse twice with deionized water. The microcapsules were then immersed in a 1.2% carboxymethyl chitosan solution obtained in step S2, and the coating was gently stirred for 12 minutes. After removal, the microcapsules were cross-linked with a 2% CaCl2 solution for 5 minutes, filtered, and the resulting encapsulated product was placed in a freeze dryer and dried at -48℃ and a vacuum of 18 Pa for 42 hours to obtain the composite probiotic agent. The undried, wet microcapsule suspension after cross-linking and curing with CaCl2 solution was taken, appropriately diluted, and measured using a laser particle size analyzer. This step involves preparing uniformly sized microdroplets using an electrostatic droplet generation method, followed by calcium ion cross-linking and layer-by-layer coating to construct a pH-responsive bilayer encapsulation structure, achieving protection of probiotics in the gastric acid environment and targeted release into the intestinal tract.

[0049] S5. Application of Compound Probiotic Agent in Corn Pulp: Select fresh corn kernels, wash and pulp them, adjusting the solids content to 12%. Place the corn pulp with a 12% solids content in a jacketed kettle, heat to 98℃, maintain for 18 minutes for sterilization, and then cool to 38℃. Add the compound probiotic agent obtained in step S4 at 0.4% of the total mass of the corn pulp with a 12% solids content, stir evenly, and ferment in a 38℃ constant temperature fermentation tank for 21 hours. After fermentation, refrigerate the product at 4℃ to obtain a flavor-improved corn pulp beverage. This step utilizes the synergistic fermentation effect of the compound probiotic agent to convert starch and protein in the corn pulp into small molecule sugars, amino acids, and flavor compounds, significantly enhancing the sweetness, milky aroma, and overall flavor profile of the beverage.

[0050] Example 4 S1. Preparation of modified sodium alginate: Weigh 30 g of sodium alginate powder and dissolve it in 500 mL of deionized water. Place the solution in a 70℃ water bath and stir at 400 r / min until completely dissolved. Add 15 mL of octenyl succinic anhydride. Monitor the pH of the reaction solution with a pH meter and adjust the pH to 9.0 with 1 mol / L NaOH solution. Maintain the pH of the system at 9.0 by adding NaOH solution dropwise during the reaction. Continue stirring for 4 hours. After the reaction is complete, pour the reaction solution into 3 times its volume of 95% ethanol to precipitate the solid. Filter the precipitate using a Buchner funnel. Wash the obtained solid three times with 70% ethanol and then transfer it to a vacuum drying oven. Dry the solid at 60℃ for 8 hours to obtain hydrophobically modified sodium alginate powder. This step introduces octenyl succinic acid groups into the sodium alginate molecular chain through esterification, enhancing the hydrophobicity and acid resistance of the material and providing a more stable wall material for subsequent embedding.

[0051] S2. Preparation of modified chitosan: Weigh 15 g of chitosan powder and add it to 300 mL of isopropanol. Stir and disperse at 500 r / min for 30 minutes. Add 10 mL of 10 M sodium hydroxide solution and alkalize in an ice-water bath for 1 hour. Then add 30 g of sodium chloroacetate and place the mixture in a 70℃ water bath, stirring continuously for 6 hours. After the reaction is complete, pour the reaction solution into 3 times its volume of 95% ethanol to precipitate, filter, wash the solid three times with 70% ethanol, and then place it in a vacuum drying oven and dry at 60℃ for 8 hours to obtain carboxymethyl chitosan powder. This step introduces carboxymethyl groups into the chitosan molecule through carboxymethylation, which significantly improves its water solubility and bioadhesion, and enhances the retention and release performance of microcapsules in the intestine.

[0052] S3. Preparation of the compound probiotic suspension: *Lactobacillus plantarum* and *Lactobacillus fermentum* were inoculated into MRS liquid medium, and *Saccharomyces cerevisiae* was inoculated into YPD liquid medium. All were incubated statically at 40℃ for 24 hours. Each bacterial suspension was centrifuged at 6000 r / min for 12 minutes, the supernatant was discarded, and the bacterial cells were resuspended in sterile physiological saline. The bacterial concentration was adjusted to 1×10⁻⁶ using a hemocytometer. 10 CFU / mL. The three bacterial suspensions were mixed at a volume ratio of 1:1:1 to obtain a compound probiotic suspension. This step yields a highly active and balanced compound probiotic, providing core live bacteria material for encapsulation.

[0053] S4. Encapsulation of the compound probiotic agent: Weigh 5 g of the modified sodium alginate powder obtained in step S1, dissolve it in 200 mL of deionized water, and stir in a 60℃ water bath until completely dissolved. Add 30 mL of the compound probiotic suspension obtained in step S3, and mix evenly with a magnetic stirrer at 500 r / min. Pump the above mixture at a flow rate of 1.0 mL / h using an electrostatic droplet generator, forming uniform microdroplets under a 12 kV voltage, and drop them into a container containing 4% CaCl2 solution (placed on a magnetic stirrer and slowly stirred at 150 r / min). After the droplets come into contact with Ca²⁺, they rapidly gel into spheres and crosslink for 20 minutes. Filter out the microcapsules with a sieve and rinse twice with deionized water. The microcapsules were then immersed in a 1.5% carboxymethyl chitosan solution obtained in step S2, and the coating was gently stirred for 15 minutes. After removal, the microcapsules were cross-linked with a 2% CaCl2 solution for 5 minutes, filtered, and the resulting encapsulated product was placed in a freeze dryer and dried at -50°C and a vacuum of 20 Pa for 48 hours to obtain the composite probiotic agent. The undried, wet microcapsule suspension after cross-linking and curing with CaCl2 solution was taken, appropriately diluted, and measured using a laser particle size analyzer. This step involves preparing uniformly sized microdroplets using an electrostatic droplet generation method, followed by calcium ion cross-linking and layer-by-layer coating to construct a pH-responsive bilayer encapsulation structure, achieving protection of probiotics in the gastric acid environment and targeted release into the intestinal tract.

[0054] S5. Application of Compound Probiotic Agent in Corn Pulp: Select fresh corn kernels, wash and pulp them, adjusting the solids content to 12%. Place the corn pulp with a 12% solids content in a jacketed kettle, heat to 100℃, maintain for 20 minutes for sterilization, and then cool to 40℃. Add the compound probiotic agent obtained in step S4 at 0.5% of the total mass of the corn pulp with a 12% solids content, stir evenly, and ferment in a 40℃ constant temperature fermentation tank for 24 hours. After fermentation, refrigerate the product at 4℃ to obtain a flavor-improved corn pulp beverage. This step utilizes the synergistic fermentation effect of the compound probiotic agent to convert starch and protein in the corn pulp into small molecule sugars, amino acids, and flavor compounds, significantly enhancing the sweetness, milky aroma, and overall flavor profile of the beverage.

[0055] Comparative Example 1: Except for step S4, which was changed to weighing 3.5 g of unmodified sodium alginate powder, all other steps were the same as in Example 1.

[0056] Comparative Example 2: Except for step S4, which omits the encapsulation step and instead uses the unencapsulated bacterial powder obtained by freeze-drying the compound probiotic suspension prepared in step S3, all other steps are the same as in Example 1.

[0057] Comparative Example 3: Except for step S4, which uses a common chitosan solution prepared in 1% acetic acid solution to coat the coating instead of carboxymethyl chitosan solution, all other steps are the same as in Example 1.

[0058] Comparative Example 4: Except for step S4, which only involves cross-linking modified sodium alginate-calcium ions to form spheres without subsequent carboxymethyl chitosan coating and secondary cross-linking to obtain monolayer sodium alginate-calcium gel microspheres, all other steps are the same as in Example 1.

[0059] Comparative Example 5: Except for step S4, where the modified sodium alginate solution is mixed with the compound probiotic suspension and then freeze-dried directly (omitting the step of adding CaCl2 solution to cross-link into spheres), all other steps are the same as in Example 1.

[0060] Comparative Example 6: Except for steps S3 and S4, only 20 ml of *Lactobacillus plantarum* (concentration 5 × 10⁻⁶) was used. 9 (CFU / mL), the remaining steps are the same as in Example 1.

[0061] Comparative Example 7: Except for steps S3 and S4, only 20 ml of brewing yeast (concentration of 1×10⁻⁶) was used. 9 (CFU / mL), the remaining steps are the same as in Example 1.

[0062] Comparative Example 8: Except for step S5, which does not involve the addition of any probiotic agent, all other steps are the same as in Example 1.

[0063] Comparative Example 9: Except for step S4, in which unmodified sodium alginate powder and unmodified chitosan solution dissolved in acetic acid were used for double-layer embedding, the other steps were the same as in Example 1.

[0064] Simulated gastric juice tolerance test The principle of viable bacteria counting was referenced in GB 4789.35-2016 National Food Safety Standard - Microbiological Examination of Food - Examination of Lactic Acid Bacteria, and the test was conducted in a simulated human stomach environment.

[0065] ① Prepare simulated gastric juice: Take 3.2 g of pepsin (activity ≥800 U / mg) and 2.0 g of NaCl, dissolve them in about 800 mL of ultrapure water, adjust the pH to 2.0±0.1 with 1 mol / L HCl, and make up to 1000 mL. Prepare fresh and use immediately.

[0066] ② Accurately weigh 0.1g of each of the following samples: Example 1, Comparative Example 1, and Comparative Example 2. All samples were prepared as the "compound probiotic agent" (microcapsule powder) in step S4. Add each sample to 10mL of sterile physiological saline and vortex to mix thoroughly. This will serve as the initial sample solution. The initial viable cell count was determined using the plate pour method and denoted as N0.

[0067] ③ Take 1 mL of the above initial sample solution and add it to 9 mL of simulated gastric fluid preheated to 37℃. Mix well and place in a 37℃ constant temperature water bath shaker, and shake at 100 r / min.

[0068] ④ Take 1 mL samples at 0 minutes (immediately after addition), 60 minutes, and 120 minutes after treatment. Immediately perform 10-fold serial dilutions with pH 7.0 phosphate buffer, selecting 2-3 suitable dilutions. Spread 0.1 mL of each dilution onto MRS (lactic acid bacteria) and YPD (yeast) agar plates, with two replicates for each dilution. Incubate the lactic acid bacteria plates anaerobically at 37°C for 48 hours, and the yeast plates at 30°C for 48 hours, then count the colonies.

[0069] ⑤ Formula for calculating survival rate: Survival rate (%) = (N t / N0)×100%, where N t The number of viable bacteria (CFU / g) after treatment for time t.

[0070] Table 1. Viable bacterial survival rate (%, mean ± standard deviation, n=3) of different samples after treatment in simulated gastric fluid (pH 2.0)

[0071] Note: ND indicates not detected. The results show that the unencapsulated probiotics were completely inactivated after 120 minutes of treatment with simulated gastric juice.

[0072] From Table 1 and Figure 1 It was found that after treatment with simulated gastric juice (pH 2.0) for 120 minutes, the survival rate of the three probiotics in Example 1 (using modified sodium alginate) remained above 80%, significantly higher than that of Comparative Example 1 (using unmodified sodium alginate, survival rate of about 30-40%) and Comparative Example 2 (without encapsulated bacterial powder, almost all of them were inactivated). This proves that octenyl succinic anhydride modification endows sodium alginate with stronger hydrophobicity and acid resistance, effectively blocking the erosion of probiotics by gastric acid, which is one of the key innovations of this invention. After treatment with simulated gastric juice for 120 minutes, the survival rate of Comparative Example 9 (traditional double-layer unmodified system) was only about 18%-25%, significantly lower than that of Example 1 (>80%), and also significantly lower than that of Comparative Example 1 (using modified shell, about 30-40%). This demonstrates that relying solely on the traditional unmodified chitosan coating is insufficient to effectively block the penetration of gastric acid into the unmodified sodium alginate core, proving the necessity of simultaneously modifying both the core and the outer shell in this invention to improve acid resistance.

[0073] Furthermore, to further verify the synergistic effect of the bilayer modified structure, this invention supplemented the investigation of the tolerance of Comparative Example 3 (modified sodium alginate core + unmodified chitosan shell) in simulated gastric fluid. The results showed that after 120 minutes of treatment, the probiotic survival rate in Comparative Example 3 was 62.5%-68.4%. This data was significantly higher than that of Comparative Example 9 (traditional unmodified bilayer, survival rate 18.6%-25.4%) and Comparative Example 1 (unmodified core + modified shell, survival rate 30.4%-41.2%), confirming that the hydrophobic barrier provided by the modified sodium alginate core is the main contributing factor to acid resistance. Meanwhile, the survival rate of Comparative Example 3 was still significantly lower than that of Example 1 (double modification, survival rate >80%). This indicates that although the unmodified chitosan shell is easily dissolved or swollen in acidic environments, resulting in a weaker protective effect on the core compared to the modified chitosan shell, it reduces the final acid resistance effect. In summary, the "octenyl succinic anhydride modified sodium alginate core" and the "carboxymethyl chitosan shell" used in this invention form a synergistic effect mechanism, which together achieves a high survival rate of probiotics in the acidic environment of the stomach.

[0074] II. Verification of the Small Intestinal Targeted Release and Adhesion Performance of Compound Probiotic Agents (1) Determination of release rate in pseudo-intestinal fluid ① Preparation of simulated intestinal fluid: Refer to the General Chapter of Part IV of the 2020 edition of the Chinese Pharmacopoeia, take 10.0 g of pancreatic enzyme (activity ≥250 U / mg) and 6.8 g of potassium dihydrogen phosphate, dissolve them in purified water, adjust the pH to 6.8±0.1 with 0.1 mol / L sodium hydroxide solution, and make up to 1000 mL.

[0075] ② Accurately weigh 0.10g of the sample from Example 1 and the sample from Comparative Example 3. The test samples are all the “compound probiotic agent” (microcapsule powder) prepared in step S4. Place them in the pretreated dialysis bags (molecular weight cutoff 8000-14000), add 5 mL of simulated intestinal fluid preheated to 37°C, and tie the bag tightly.

[0076] ③ Immerse the dialysis bag completely in 200 mL of simulated intestinal fluid release medium at 37°C, and place it in a 37°C constant temperature water bath shaker, and shake slowly at 50 rpm.

[0077] ④ At time points of 0, 1, 2, 4, 6, and 8 hours, remove the dialysis bag, cut open the bag, aspirate the liquid inside, and determine the number of viable bacteria released. Calculate the cumulative release rate (%) = (cumulative number of viable bacteria released / total number of viable bacteria in the sample) × 100%.

[0078] (2) In vitro mucosal adhesion force measurement ① Take fresh pig small intestine mucosa, wash it with physiological saline, and then lay it flat on a glass slide.

[0079] ② Sprinkle the powder from the sample group of Example 1 and the sample group of Comparative Example 3 evenly on the moist mucosal surface and let it stand for 5 minutes. Among them, the test samples are all "compound probiotics" (microcapsule powder) prepared in step S4.

[0080] ③ Rinse the mucosal surface slowly and at a constant speed for 30 seconds with a saline solution containing 0.1% (w / v) trypan blue to remove any unadhered particles.

[0081] ④ Observe and photograph under a microscope, and use image analysis software to calculate the percentage of the area covered by the blue microcapsules remaining on the mucosal surface (because chitosan can adsorb trypan blue), and use this to semi-quantitatively evaluate the adhesion (see Table 4 for the scoring criteria).

[0082] Table 2. Cumulative release rate of viable bacteria (%, mean ± standard deviation, n=3) of different samples in simulated intestinal fluid (pH 6.8)

[0083] Table 3 Evaluation of in vitro mucosal adhesion of different samples

[0084] Table 4 Relative Adhesion Rating Criteria

[0085] As shown in Table 2, in simulated intestinal fluid, the probiotics released from Example 1 (carboxymethyl chitosan coating) were released faster and more completely, with a release rate of 98.1% after 8 hours, significantly higher than that of Comparative Example 3 (ordinary chitosan coating, approximately 79%). Simultaneously, its in vitro mucosal adhesion score was also significantly higher. This indicates that carboxymethylation modification greatly improves the water solubility and bioadhesion of chitosan, enabling the encapsulated probiotics to be efficiently released and easily retained in the intestinal environment, achieving targeted intestinal delivery, which is another key innovation of this invention. Comparative Example 9 (traditional bilayer system) showed a release rate of only 73.5% in simulated intestinal fluid, far lower than that of Example 1 (98.1%). This is mainly because unmodified chitosan has poor solubility in the neutral pH environment of the intestine, hindering the disintegration of the microcapsules and the release of the internal probiotics. This data comparison further demonstrates the crucial role of using carboxymethyl chitosan-modified shells for achieving efficient targeted intestinal release.

[0086] It is worth noting that the inventors discovered that simply using a single modification (such as Comparative Example 1 or Comparative Example 3) cannot simultaneously achieve both acid resistance and enteric solubility. In particular, Comparative Example 9 (the traditional sodium alginate-chitosan system) performed poorly in both gastric juice survival rate and intestinal juice release rate, both significantly lower than Example 1 of this invention. This strongly demonstrates that the dual-modified pH-sensitive bilayer structure constructed by the present invention, using an octenyl succinic anhydride-modified sodium alginate core and a carboxymethyl chitosan shell, has a significant synergistic effect in improving probiotic survival rate and achieving targeted intestinal release.

[0087] III. Verification of the in vivo intestinal colonization effect of compound probiotics (1) Experimental animals and grouping: Sixty healthy male Balb / c mice, 6 weeks old, were randomly divided into 4 groups (Example 1 group, Comparative Example 4 group, Comparative Example 5 group, and saline blank control group), with 15 mice in each group. They were housed separately under standard conditions and had free access to food and water. All experimental animal operations were performed in accordance with the "Guidelines for the Care and Use of Laboratory Animals".

[0088] (2) Experimental method: ① Gavage treatment: Gavage was administered at a fixed time each day for 7 consecutive days. Mice in Example 1 group, Comparative Example 4 group, and Comparative Example 5 group were gavage with an equal amount of live bacteria (5 × 10⁻⁶). 9 The corresponding sample suspension (prepared with physiological saline) of CFU / animal / day was used. The test samples were all "compound probiotics" (microcapsule powder) prepared in step S4. The blank control group was given an equal volume of sterile physiological saline by gavage.

[0089] ② Fecal sample collection and processing: Fresh feces were aseptically collected from each group of mice before the start of gavage (day 0), on the day after the last gavage (day 7), and on day 7 after the cessation of gavage (day 14). Five mice were randomly selected from each group at each time point, and their feces were collected as independent samples (n=5).

[0090] ③ Quantitative analysis of intestinal flora: Weigh 0.10 g of feces, add 0.90 mL of sterile physiological saline, vortex to prepare a homogenate, and perform a 10-fold serial dilution. Select an appropriate dilution and spread it onto MRS selective agar plates containing vancomycin (20 μg / mL) and bromocresol purple. This culture medium can effectively inhibit the growth of non-lactobacterial bacteria such as streptococci and enterococci, and is used for selective counting of the total number of lactobacilli. Because the gavage dose in this experiment was as high as 5 × 10⁻⁶ g / mL... 9CFU / mouse, with *Lactobacillus plantarum* and *Lactobacillus fermentum* being the main components administered via gavage. The significantly increased number of *Lactobacillus* species detected can be mainly attributed to the successful colonization of exogenous probiotics. Simultaneously, *Saccharomyces cerevisiae* (a rare endogenous yeast in the mouse gut, considered an exogenous indicator) was counted using YPD selective agar plates containing chloramphenicol. Plates were cultured under appropriate conditions and counted, with results expressed as log10 CFU / g of colony-forming units per gram of wet feces.

[0091] Table 5. Changes in the number of Lactobacillus and Saccharomyces cerevisiae in mouse feces after gavage in different samples (log10 CFU / g, mean ± standard deviation, n=5)

[0092] From Table 5 and Figure 3 Before gavage (day 0), the baseline level of *Lactobacillus* in the mouse intestine was 5.2 log10 CFU / g. Seven days after gavage, the number of *Lactobacillus* in the feces of mice in Example 1 group surged to 8.5 log10 CFU / g, an increase of more than three orders of magnitude compared to the baseline level, and significantly higher than the control group. Considering that the number of endogenous *Lactobacillus* in mice is relatively stable, this significant increase in number mainly comes from the successful colonization of exogenously added *Lactobacillus plantarum* and *Lactobacillus fermentum*. One week after stopping gavage (day 14), the number of *Lactobacillus* in the intestine of mice in Example 1 group remained at a high level of 7.9 log10 CFU / g, showing good colonization ability. Notably, the number of *Saccharomyces cerevisiae* in Example 1 group was 3.2 log10 CFU / g, significantly higher than that in control group 5 (not detected) and control group 4 (not detected). Although *Saccharomyces cerevisiae* is generally considered a transient bacterium and difficult to colonize long-term, the excellent bioadhesion of the microcapsules of this invention enabled it to achieve a significant retention effect in the intestine. This indicates that the modified chitosan shell enhances the adhesion of the microcapsules to the intestinal mucosa and prolongs the residence time of Saccharomyces cerevisiae in the intestine, making it still detectable 7 days after the cessation of gavage. This is of great significance for the sustained exercise of the metabolic regulatory function of probiotics. Figure 3 The stability of the microcapsules in simulated gastric juice and their disintegration behavior in simulated intestinal juice were visually demonstrated, confirming their pH sensitivity. This proves that the pH-sensitive bilayer microcapsule structure constructed in this invention can most effectively protect probiotics as they pass through the stomach and are released and colonized at specific points in the intestine, exhibiting excellent in vivo targeted delivery and sustained colonization effects.

[0093] IV. Verification of the Application Effect of Compound Probiotic Agent in Enhancing the Flavor of Corn Pulp (1) Detection of key flavor compounds: Headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) was used for determination. 5 mL of the fermented sample (all test samples were corn pulp beverages after fermentation in step S5) was placed in a 20 mL headspace vial, 1 g NaCl was added, and an activated 50 / 30 μm DVB / CAR / PDMS extraction head was inserted. Adsorption was performed at 60℃ for 30 minutes, followed by desorption. GC-MS conditions: DB-WAX capillary column (60 m × 0.25 mm × 0.25 μm); injection port temperature 250℃; temperature program: initial temperature 40℃ held for 3 min, increased to 150℃ at 5℃ / min, then increased to 230℃ at 10℃ / min, held for 5 min; mass spectrometry scan range m / z 35-350. Qualitative analysis was performed by standard comparison and spectral library search. Semi-quantitative analysis of the main flavor compounds was conducted using the internal standard method (with 2-octanol as the internal standard).

[0094] (2) Sensory evaluation: Referring to GB / T 10220-2012 "General Methodology of Sensory Analysis" and GB / T 16861-1997 "General Series Test Methods and Results of Sensory Analysis", a group of 10 trained sensory evaluators scored the color, aroma, taste, mouthfeel and overall acceptability of the samples. A 5-point Likert scale (1 - very dislike, 5 - very like) was used.

[0095] Table 6 Comparison of the content of major flavor compounds

[0096] Table 7 Average Sensory Evaluation Scores

[0097] From Table 6 and Figure 4 It can be seen that Example 1 (compound microbial fermentation) produced the richest spectrum of flavor compounds, including lactic acid and acetic acid contributed by lactic acid bacteria (providing sourness and aroma), ethyl acetate and phenylethanol contributed by yeast (providing fruity and floral aromas), and acetoin (providing milky and creamy aromas) enhanced by the synergistic effect of both. In contrast, Comparative Examples 6 and 7, due to their single microbial strains, had fewer types and amounts of flavor compounds than Example 1. (See Table 7 and...) Figure 5 As can be seen, the sensory evaluation results are consistent with the instrumental analysis. The scores of Example 1 group in aroma, taste, and overall acceptability are significantly higher than all comparative groups. This fully demonstrates that the combination of *Lactobacillus plantarum*, *Lactobacillus fermentum*, and *Saccharomyces cerevisiae* produces a synergistic effect, which can comprehensively improve the flavor and taste of corn pulp, and is the core application value of this invention.

[0098] V. Characterization of the hydrophobicity of modified sodium alginate (contact angle measurement) Experimental Principle: Contact angle is an important indicator of the wettability of a material surface. The larger the contact angle, the stronger the hydrophobicity of the material surface. This invention modifies sodium alginate with octenyl succinic anhydride to introduce hydrophobic groups, aiming to improve its acid resistance. The modification effect can be visually verified through contact angle testing.

[0099] Experimental Methods: 1 g each of unmodified sodium alginate (control group) and modified sodium alginate prepared in Example 1 (Example 1 group) were weighed and dissolved in 50 mL of deionized water. The solution was magnetically stirred until completely dissolved to prepare a 2% (w / v) solution. The solution was poured into a polytetrafluoroethylene mold and cast into a film at room temperature. After natural drying, the film was peeled off. The water contact angle of the film surface was measured at room temperature using a contact angle meter and the seat drop method. Five different points were selected for measurement for each sample, and the average value was taken.

[0100] Table 8. Results of water contact angle measurement of sodium alginate films before and after modification.

[0101] Table 8 shows that the unmodified sodium alginate film has a small water contact angle (32.5°), exhibiting strong hydrophilicity. This is because the sodium alginate molecular chain contains a large number of hydrophilic hydroxyl and carboxyl groups. After modification with octenyl succinic anhydride, the contact angle of the sodium alginate film significantly increased to 78.4° (P<0.05), indicating that octenyl succinic anhydride successfully introduced hydrophobic long-chain alkyl groups, significantly improving the hydrophobicity of the material. This enhanced hydrophobicity helps the microcapsules shrink the gel pores in an acidic environment, blocking the penetration of H⁺ and pepsin in gastric acid, thereby effectively protecting the encapsulated probiotics.

[0102] VI. Characterization of the water solubility of modified chitosan (solubility determination) Experimental Principle: Ordinary chitosan is only soluble in acidic solutions and precipitates in neutral or weakly alkaline environments, limiting its targeted release in the intestine (neutral environment). This invention aims to disrupt the hydrogen bonds between chitosan molecules and introduce hydrophilic groups through carboxymethylation modification, thereby improving its solubility in water.

[0103] Experimental Method: 0.5 g each of unmodified chitosan (control group, degree of deacetylation ≥90%) and carboxymethyl chitosan prepared in Example 1 (Example 1 group) were weighed and added to beakers containing 50 mL of deionized water. The mixtures were magnetically stirred for 30 minutes, and the dissolution was observed. The pH of the solution was then adjusted to 7.0 with 1 mol / L NaOH solution, and stirring continued while observing for any precipitation. After centrifugation, the supernatant was dried, weighed, and the solubility was calculated.

[0104] Table 9. Comparison of solubility of chitosan before and after modification at different pH values.

[0105] Experimental results show that unmodified chitosan swells in water but is difficult to dissolve, and precipitates under neutral conditions. In contrast, the carboxymethyl chitosan prepared in this invention exhibits excellent solubility in water and remains clear and transparent even in a neutral environment of pH 7.0. This characteristic verifies the success of carboxymethylation modification, enabling the modified chitosan, as a microcapsule shell material, to rapidly swell and release probiotics within the neutral environment of the intestine, achieving a "targeted intestinal release" function.

[0106] Based on experimental data, the inventors hypothesize that the mechanism of action of this compound probiotic agent is as follows: The acid-resistant / enteric-coating mechanism of pH-sensitive bilayer microcapsules: The octenyl succinic anhydride (OSA) modified sodium alginate core shrinks and reduces pore size in an environment with hydrophobic octenyl succinic acid groups, effectively blocking H⁺ and pepsin penetration, thus protecting probiotics; while the carboxymethyl chitosan shell introduces hydrophilic groups through carboxymethylation, rapidly swelling and disintegrating in intestinal fluid (pH 6.8) to achieve targeted release. The synergistic effect of the bilayer structure is far superior to that of single modification, forming a pH-responsive switch of "acid contraction-alkali disintegration".

[0107] The synergistic flavor enhancement mechanism of the compound microbial strains: *Lactobacillus plantarum* and *Lactobacillus fermentum* produce lactic acid and acetic acid through glycolysis, while *Saccharomyces cerevisiae* contributes esters / alcohols such as ethyl acetate and phenylethanol (imbuing fruit and floral aromas). Through cross-metabolic interactions (organic acids produced by lactic acid bacteria provide precursors for yeast, while yeast metabolites conversely promote lactic acid bacteria growth), the three strains significantly enhance acetoin, ultimately improving the conversion efficiency of starch / protein in corn pulp into small-molecule flavor compounds, transforming the taste from "rough and simple" to "rich and mellow." Furthermore, in vivo colonization data (maintaining a high level even on day 14) further indicates that the bioadhesiveness of modified chitosan prolongs the retention time of the microorganisms in the intestine, enhancing overall probiotic function.

[0108] The above-described embodiments are merely illustrative of specific implementations of the present invention, and while the descriptions are detailed, they should not be construed as limiting the scope of protection of the present invention. It should be noted that for those skilled in the art, any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the scope of protection of the present invention.

Claims

1. A compound probiotic agent, characterized in that, The probiotic agent has a pH-sensitive bilayer microcapsule structure, including a core and a shell; The core is formed by encapsulating probiotics with modified sodium alginate, and the outer shell is formed by coating the surface of the core with modified chitosan; The compound probiotics include Lactobacillus plantarum, Lactobacillus fermentum, and Saccharomyces cerevisiae.

2. The compound probiotic agent according to claim 1, characterized in that, The modified sodium alginate is sodium alginate modified with octenyl succinic anhydride.

3. The compound probiotic agent according to claim 1, characterized in that, The modified chitosan is carboxymethyl chitosan.

4. The compound probiotic agent according to claim 1, characterized in that, The volume ratio of *Lactobacillus plantarum*, *Lactobacillus fermentum*, and *Saccharomyces cerevisiae* is 1:(0.5-2):(0.5-2).

5. A method for preparing the compound probiotic agent according to any one of claims 1-4, characterized in that, Includes the following steps: Modified sodium alginate was prepared, modified chitosan was prepared under alkaline conditions, a compound probiotic suspension was prepared, and a compound probiotic agent was prepared by calcium ion crosslinking and coating encapsulation.

6. The preparation method according to claim 5, characterized in that, The preparation of modified sodium alginate includes dissolving sodium alginate in deionized water, adding octenyl succinic anhydride, reacting under alkaline conditions, and then precipitating and drying.

7. The preparation method according to claim 5, characterized in that, The preparation of modified chitosan includes dispersing chitosan in isopropanol, adding sodium chloroacetate under alkaline conditions, and then precipitating and drying the product after the reaction.

8. The preparation method according to claim 5, characterized in that, The process of cross-linking and coating with calcium ions includes mixing a modified sodium alginate solution with a compound probiotic suspension, adding a calcium salt solution for cross-linking, coating with a modified chitosan solution, and then drying.

9. The application of the compound probiotic agent according to any one of claims 1-4 in improving the flavor of corn pulp.

10. The application according to claim 9, characterized in that, The method of this application is as follows: select fresh corn kernels or soaked dried corn kernels, wash them and then pulp them to obtain corn pulp. Adjust the solid content of the corn pulp to 10-15%, sterilize it, and then inoculate it with the compound probiotic agent described in any one of claims 1-4 for fermentation. The fermentation temperature is 35-40℃ and the fermentation time is 12-24 hours.

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