Preparation process of functional soybean milk powder with sleep improving effect

Abstract

The invention belongs to the field of functional food processing, and provides a preparation process of functional soybean milk powder with a sleep improving effect, which adopts a five-step linkage design of germinated soybean enzymolysis, lactobacillus plantarum fermentation, in-situ calcium nucleation, isoelectric point adsorption, exopolysaccharide stabilization and spray drying. Layered coating of the fermentation system autogenous exopolysaccharide and the fermentation modified soybean protein on the organic acid calcium microcrystal core is realized through pH gradient dynamic regulation and control, and a core-protein middle layer-polysaccharide outer layer ternary in-situ composite microcapsule structure is constructed. The multi-target synergy that the gamma-aminobutyric acid is enriched to 150-300 mg / 100 g, the isoflavone aglycone rate reaches 70-85% and the microcapsule D50 is controlled to be 5-80 microns is achieved, the three technical problems that high-solid-content low-viscosity spraying and strong interaction conflict exists, the dry powder flowability and core-shell layer stability conflict exists, and a narrow pH window and amplification tolerance are opposite are solved, and the method has wide functional food application value.

Description

Technical Field

[0001] This invention belongs to the field of functional food processing and provides a process for preparing functional soy milk powder with sleep-improving effects. Background Technology

[0002] With the fast pace of modern life and increasing work pressure, sleep disorders have become a significant public health issue, and functional foods, as a non-pharmaceutical intervention, have received widespread attention. Soy milk powder, as an important form of plant-based protein beverage, possesses unique advantages in functional modification. Through biotransformation technologies such as germination and fermentation, the content of sleep-aiding active ingredients such as γ-aminobutyric acid (GABA) and isoflavone aglycones can be significantly increased. However, achieving efficient enrichment of functional ingredients is only the first step. Solving the processing suitability challenge of calcium-fortified soy milk powder while ensuring the stability of active ingredients—namely, maintaining sprayable low-viscosity rheological properties in a high-solids-content slurry state, ensuring good flowability and rehydration of the dry powder, and maintaining the activity of functional ingredients and the integrity of the microcapsule structure during storage—is the core requirement for the current development of functional soy milk powder products. Meeting these complex performance requirements is crucial for promoting the large-scale production of functional soy milk powder from the laboratory, expanding its application scope in special dietary foods and functional beverages, and enhancing product market competitiveness. It is also a key breakthrough for the food industry to achieve a synergistic improvement in nutritional health and processing efficiency.

[0003] Currently, the functionalization of soy milk powder mainly relies on the combined germination-fermentation technology to increase the content of active ingredients, but significant technical bottlenecks exist when transitioning to high-solids spray drying processes. For example, Chinese patent CN108713599A discloses a method for preparing selenium-enriched germinated brown rice-based mung bean milk powder rich in γ-aminobutyric acid (GABA), but it suffers from drawbacks such as a sharp increase in system viscosity due to protein denaturation after fermentation, large particle aggregation caused by direct interaction between calcium salts and proteins during calcium fortification, and poor powder flowability and easy hygroscopic agglomeration after spray drying. The fundamental problem with existing technologies lies in the failure to achieve an integrated design of "fermentation functionalization" and "in-situ microencapsulation," leading to an irreconcilable contradiction between the requirements of high-solids, low-viscosity spray drying and the strong protein-calcium-polysaccharide interactions caused by high active ingredients / high calcium density. Simultaneously, the need for optimized dry powder flowability conflicts with the requirement for dense core-shell structure stability. Furthermore, there is a conflict between the precise pH-nucleation window control required for batch-to-batch functional consistency and the wide operational tolerance required for industrial-scale processing. These intertwined technical challenges severely restrict the industrialization process of functional soy milk powder. Summary of the Invention

[0004] The purpose of this invention is to provide a preparation process for functional soy milk powder with sleep-improving effects, addressing three major challenges in current soy milk powder fermentation-in-situ nucleation-ternary composite microcapsule processes: the contradiction between high-solids-content, low-viscosity spraying and high-activity / high-calcium nucleation density; the conflict between dry powder flowability and the stability of dense protein / polysaccharide-calcium core-shell layer; and the conflict between the narrow pH-nucleation window required for batch-to-batch functional consistency and the tolerance for scale-up processing.

[0005] This invention employs a synergistic strategy of "in-situ layered coating of self-generated dual-wall materials in a fermentation system" to overcome the aforementioned technical challenges. Through dynamic pH gradient control, *Lactobacillus plantarum* simultaneously produces two types of wall materials during fermentation: fermentation-modified soybean protein and extracellular polysaccharides. After calcium nucleation, ternary composite microcapsules are constructed sequentially using the isoelectric point electrostatic adsorption of proteins and the steric hindrance stabilization mechanism of polysaccharides. This achieves a synergistic design of "one fermentation system, zero exogenous wall materials, and dual stabilization mechanisms." This not only solves the aggregation control problem of high-calcium systems, allowing high-solids slurries to still be sprayed at low viscosity, but also improves the moisture resistance and flowability of the dry powder through core-shell densification. Simultaneously, precise pH segmentation control expands the time window for the key nucleation-coating steps from the traditional 1-2 minutes to 5-20 minutes, significantly improving the operability and batch stability for industrial scale-up. Overall, it exhibits a systemic improvement effect far exceeding the superposition of single technologies.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A process for preparing functional soy milk powder with sleep-improving effects includes the following steps:

[0008] S1: Pretreatment and enzymatic hydrolysis: Soak soybeans and then germinate them to obtain germinated soybean enzymatic hydrolysate; heat the hydrolysate to 90-95℃ and hold for 5-15 minutes to inactivate the protease; then pasteurize at 75-85℃ for 15-30 minutes.

[0009] S2: Fermentation and enrichment: After cooling the enzymatic hydrolysate obtained in step S1 to 32-37℃, inoculate it with Lactobacillus plantarum for fermentation at an inoculation amount of 2-4% (v / v). Ferment at 32-37℃ for 12-24 h. Add lactic acid or citric acid online to decrease the pH from 6.2 to 5.2 at a rate of 0.01-0.08 pH / min and maintain it until step S3 is performed to obtain a fermentation suspension.

[0010] S3: In-situ calcium nucleation: Add an organic acid calcium solution dropwise to the fermentation suspension obtained in step S2 to generate organic acid calcium microcrystal nuclei in situ within the system, thus obtaining a composite dispersion containing organic acid calcium microcrystal nuclei;

[0011] S4: Protein isoelectric point adsorption and stabilization: Adjust the pH of the composite dispersion obtained in step S3 to near the isoelectric point so that fermented modified soybean protein adsorbs and coats the microcrystal core. Then adjust the pH and age it to promote the construction of a stable layer of self-generated extracellular polysaccharide in the same fermentation system on the outer layer, and obtain a ternary in-situ composite microcapsule dispersion with microcrystal core, protein intermediate layer and extracellular polysaccharide outer layer.

[0012] S5: Concentration and spray drying: The composite microcapsule dispersion obtained in step S4 is concentrated and then spray dried to obtain the finished soy milk powder with ternary in-situ composite microcapsules as the basic unit.

[0013] Furthermore, in step S1, the soybeans are soaked at a temperature of 20-25℃ for 8-12 hours and germinated at a temperature of 26-30℃ for 24-36 hours, with an average sprout length of 1-3 mm.

[0014] Furthermore, in step S1, alkaline protease, neutral protease, or papain are added at pH 7.0-7.4 and temperature 50-55℃ for 45-75 min to hydrolyze the protein to a degree of hydrolysis (DH) of 4-10%, and the solids mass fraction in the solution is adjusted to 8-12%.

[0015] Furthermore, the alkaline protease is Subtilisin.

[0016] Furthermore, in step S2, lactic acid or citric acid is added online to decrease the pH from 6.2 to 5.2 at a rate of 0.01-0.08 pH / min and maintain this pH until step S3 is performed.

[0017] Furthermore, the pH control employs a closed-loop linkage between an online pH electrode and a metering pump, with a pH decrease rate of 0.03-0.05 pH / min and an endpoint pH value of 5.1-5.3.

[0018] Furthermore, in step S3, the system shear rate is 30-300 s under pH conditions of 5.0-5.4. -1 Add an aqueous solution or aqueous suspension of calcium lactate, calcium citrate, or calcium malate dropwise at a rate of 0.1-0.5 mL / min. -1 ·L -1 The calcium ion concentration in the system was adjusted to 8-60 mM and maintained for 5-20 min, while the system temperature was kept at 20-35℃ during the dropwise addition.

[0019] Further, in step S4, the pH of the composite dispersion is decreased from 5.0-5.4 to 4.6-4.8 within 3-10 min and maintained for 6-30 min, then the pH is adjusted back to 5.4-5.8 using sodium bicarbonate or ammonium bicarbonate at a shear rate of 10-50 s. -1 Aging under these conditions for 5-20 minutes.

[0020] Furthermore, in step S5, the material is concentrated at an outlet temperature of 20-45℃ to adjust the solid content in the concentrated liquid to 30-42%; spray drying is then performed at an inlet air temperature of 150-200℃, an outlet air temperature of 75-100℃, and an inlet temperature of 20-35℃.

[0021] As a core concept of this invention, it employs a system-integrated process of germination-enzymatic hydrolysis-pH gradient-controlled fermentation-in-situ calcium nucleation-isoelectric point adsorption-polysaccharide stabilization-spray drying, primarily used to enhance the sleep-improving function and processing suitability of soy milk powder. During the germination stage, endogenous glutamate decarboxylase is activated and anti-nutritional factors are degraded, laying a biochemical foundation for subsequent γ-aminobutyric acid (GABA) enrichment. Moderate enzymatic hydrolysis controls the degree of hydrolysis of soybean protein at 4-10%, releasing small peptides to improve the availability of the fermentation substrate while avoiding excessive hydrolysis that leads to loss of gel performance. Dynamic pH gradient control is a key innovation of this invention. By slowly lowering the pH of the fermentation system from 6.2 to 5.2 at a rate of 0.01-0.08 pH / min, the fermentation time is extended, promoting GABA accumulation and isoflavone aglycone conversion, while simultaneously inducing *Lactobacillus plantarum* to continuously secrete extracellular polysaccharides. This achieves an optimal encapsulation ratio of 1.5-2.5:1 for polysaccharides to protein, providing sufficient endogenous wall material for subsequent in-situ microencapsulation. In-situ calcium nucleation involves the dropwise addition of organic calcium acid at pH 5.0-5.4 under moderate shear conditions. The fermentation system's built-in protein-polysaccharide dual stabilizer inhibits excessive calcium nucleus growth, resulting in a controllable particle size distribution of 5-80 μm. The isoelectric point adsorption step rapidly lowers the pH to the protein's isoelectric point of 4.6-4.8, utilizing electrostatic neutralization to tightly adsorb the fermented modified soybean protein onto the calcium nucleus surface, forming the first barrier. Subsequently, the pH is adjusted back to 5.4-5.8, and low-shear aging is applied, prompting the negatively charged extracellular polysaccharide to construct a second stabilizing layer on the outer layer through hydrogen bonding and steric hindrance. Ultimately, a ternary composite structure of organic calcium acid core, protein intermediate layer, and polysaccharide shell is formed. This dual-layer coating strategy not only effectively shields the interaction between calcium ions and residual proteins, reducing the viscosity of high-solids slurry by 40-60% to meet spraying requirements, but also significantly improves the flowability and moisture resistance of dry powder by blocking the diffusion of moisture to the calcium core through a dense core-shell layer. At the same time, segmented pH control widens the critical operation window of nucleation-coating from 1-2 minutes to 5-20 minutes, greatly improving the feasibility of industrial scale-up and batch stability.

[0022] This invention also discloses a functional soy milk powder with sleep-improving effects. The soy milk powder includes a ternary in-situ composite microcapsule, wherein the microcapsule is composed of an organic acid calcium microcrystal core, a fermented modified soybean protein intermediate layer, and a self-generated extracellular polysaccharide shell of Lactobacillus plantarum from the same fermentation system.

[0023] The wall material is composed of extracellular polysaccharides generated by the fermentation system and fermentation-modified soybean protein, without the need to add exogenous wall materials;

[0024] The microcapsules have a D50 of 5-80 μm;

[0025] The soy milk powder contains 150-300 mg / 100 g of γ-aminobutyric acid and has an isoflavone aglycone conversion rate of 70-85%.

[0026] The mass ratio of the extracellular polysaccharide to the fermented modified soybean protein is 1.5-2.5:1 on a dry basis, and the mass ratio of the organic acid calcium core to the wall material is 0.35-0.50:1.

[0027] As another concept of this invention, the functional soy milk powder product provided by this invention adopts a structural design with ternary in-situ composite microcapsules as the basic unit, mainly used to enhance the product's sleep-improving function, processing suitability, and storage stability. The ternary composite microcapsules in the product consist of an organic acid calcium microcrystalline core, a fermented modified soybean protein intermediate layer, and a self-generated extracellular polysaccharide shell from *Lactobacillus plantarum*. This "homogeneous double-wall material with zero external addition" design avoids the increased cost and dilution of functional ingredients caused by the addition of maltodextrin or gum arabic required in traditional microencapsulation. The organic acid calcium microcrystalline core provides high-quality calcium fortification; the selection of organic acid calcium salts such as calcium lactate, calcium citrate, or calcium malate ensures calcium bioavailability, and their organic acid anions can also act as flavor modifiers to improve the beany taste. The fermented modified soybean protein intermediate layer tightly coats the calcium core through isoelectric point electrostatic adsorption. Utilizing the near-zero net charge of proteins at pH 4.6-4.8, maximum adsorption efficiency is achieved. This protein membrane not only physically isolates calcium ions from direct contact with the surrounding protein but also exhibits better film-forming properties and density due to the increased flexibility of the protein after fermentation modification. The extracellular polysaccharide shell is secreted by *Lactobacillus plantarum* during pH gradient-regulated fermentation. The long polysaccharide chains adsorb onto the protein layer surface via hydrogen bonds and extend into the aqueous phase, forming a steric hindrance layer. This effectively prevents the aggregation of microcapsules caused by shearing and heating during concentration and drying. Simultaneously, the hydrophilicity of the polysaccharide shell in the dry powder state allows it to adsorb a small amount of moisture, forming a "first line of defense" to protect the inner calcium core and protein from humidity fluctuations. The product contains 150-300 mg / 100 g of γ-aminobutyric acid (GABA), a range that satisfies the effective dosage required for sleep aid while avoiding potential safety concerns associated with excessively high doses. The isoflavone aglycone rate reaches 70-85%, with the bioavailability of isoflavones in aglycone form being 3-5 times higher than that of glycosides. The microcapsule D50 is controlled at 5-80 μm, ensuring good flowability of the dry powder. The extracellular polysaccharide to protein ratio of 1.5-2.5:1 is precisely achieved through pH gradient-controlled fermentation, ensuring sufficient polysaccharide to form an effective spatially stable layer while avoiding excessive polysaccharide leading to excessively high system viscosity. The organic acid calcium core to wall material ratio of 0.35-0.50:1 balances calcium fortification requirements with encapsulation efficiency.

[0028] In the ternary composite microcapsule system of this invention, fermented modified soybean protein and *Lactobacillus plantarum* self-generated extracellular polysaccharides each play their respective roles and work synergistically. Fermented modified soybean protein primarily provides a dense electrostatic adsorption layer. After fermentation controlled by a pH gradient, the protein is partially hydrolyzed into small peptides with an extended conformation. Near the isoelectric point, due to electrostatic neutralization, it rapidly adsorbs onto the surface of negatively charged organic acid calcium microcrystals, forming a dense protein film approximately 50-200 nm thick. This protein film is further densified through hydrophobic interactions and calcium bridge cross-linking, effectively shielding calcium ions from diffusing outwards and preventing external proteins from approaching inwards, thereby severing the pathway of strong calcium-protein interactions. This is the core microscopic mechanism for reducing the viscosity of high-solids slurries. Extracellular polysaccharides primarily provide a flexible steric stabilizing layer. *Lactobacillus plantarum* continuously secretes dextran or fructan-type extracellular polysaccharides as the pH slowly decreases from 6.2 to 5.2. These polysaccharides typically have a molecular weight of 10... 5 -10 6 The long-chain structure of the extracellular polysaccharide facilitates adsorption onto the protein layer surface via hydrogen bonds and extends into the aqueous phase, forming a "brush" conformation. When two microcapsules approach each other, the polysaccharide chains overlap, triggering osmotic repulsion and effectively preventing aggregation. The extracellular polysaccharide contributes to the high solids content and low viscosity by its excellent shear-thinning properties. Under the high shear of spraying, the polysaccharide chains orient themselves, reducing viscosity, while restoring the network upon standing, providing suspension stability. Regarding the synergy between dry powder flowability and core-shell stability, the density of the protein layer ensures that the calcium core does not undergo phase transitions or crystal growth during drying, while the hydrophilicity of the polysaccharide shell preferentially adsorbs trace amounts of moisture from the environment, forming a monolayer of water. This water film acts as both a "lubricant" to improve powder flowability and a "sacrificial layer" to protect the inner protein-calcium structure from humidity shock. The synergistic effect of the two types of wall materials is also reflected in the broadening of the pH control window. Protein adsorption reaches its maximum efficiency at pH 4.6-4.8, but the time window is only 1-2 minutes, while polysaccharide adsorption proceeds slowly at pH 5.4-5.8 under low shear aging conditions, which can last for 5-20 minutes. This two-step segmented operation avoids the harsh dependence on a single pH point. This time-sequential decoupling strategy significantly improves the operational tolerance for industrial scale-up. In summary, fermented modified soybean protein constructs an "internal defense" through electrostatic adsorption and densification to sever strong interactions, while extracellular polysaccharides construct an "external defense" through steric hindrance and shear thinning to provide rheological optimization. Both are generated in situ within the same fermentation system and play their roles sequentially, achieving a full-chain synergy from molecular-level interaction regulation to macroscopic-level improvement in processing and storage stability. This dual-wall material strategy, which combines internal and external treatments and rigidity and flexibility, is the key to this invention's breakthrough of traditional technical bottlenecks.

[0029] (3) Beneficial technical effects

[0030] 1. This technology integrates fermentation functionalization with in-situ microencapsulation, overcoming the bottlenecks of traditional step-by-step processes. Through dynamic pH gradient control, *Lactobacillus plantarum* simultaneously produces two types of wall materials during fermentation: fermentation-modified soybean protein and extracellular polysaccharides. This eliminates the need for traditional wall materials such as maltodextrin or gum arabic, reducing costs by 15-25% and avoiding the decrease in functional component concentration caused by dilution from exogenous wall materials. Furthermore, it simplifies the process, reduces independent unit operations in microcapsule preparation, and improves overall production efficiency by over 30%.

[0031] 2. Overcoming the contradiction between high-solids-content, low-viscosity spray drying and strong calcium-protein interaction, significantly improving processability. Through a dual stabilization mechanism of protein isoelectric point electrostatic adsorption and polysaccharide steric hindrance, the solids concentration can still be increased to 30-42% in high-calcium systems (calcium ion concentration 8-60 mM), while the viscosity is reduced by 40-60% compared to traditional processes. This allows high-solids-content slurries to meet spray drying requirements, reducing spray energy consumption by 20-30%. At the same time, the ternary core-shell structure effectively inhibits calcium salt crystallization and protein aggregation during drying. The flowability index of the finished powder is increased from 45-55 in traditional processes to 75-85 (Carr index method), and the moisture resistance is improved by more than 50%.

[0032] 3. Expanding the pH-nucleation operation window improves the feasibility of industrial scale-up and batch stability. Traditional in-situ nucleation requires precise control at a single pH point (usually 4.8-5.0) for 1-2 minutes. During scale-up, pH fluctuations can easily lead to uncontrolled particle size distribution. This invention decouples protein adsorption (pH 4.6-4.8, 6-30 min) and polysaccharide stabilization (pH 5.4-5.8, 5-20 min) through a segmented pH control strategy, expanding the overall operation window to 15-50 minutes and increasing the tolerance to pH fluctuations from ±0.05 to ±0.15. During 10-ton pilot-scale scale-up, the batch variation coefficient of microcapsule D50 decreased from 18-25% in the traditional process to 5-8%, significantly improving batch-to-batch functional consistency.

[0033] 4. Highly enriches sleep-improving active ingredients, significantly enhancing functionality. Germination activates endogenous glutamate decarboxylase, pH gradient regulation prolongs fermentation time and optimizes the metabolic pathways of the strain, resulting in a γ-aminobutyric acid (GABA) content of 150-300 mg / 100g, 2-3 times higher than traditional fermented soy milk powder. Simultaneously, fermentation promotes isoflavone aglycone conversion, achieving an aglycone rate of 70-85%, with bioavailability 3-5 times higher than glycosides. Animal sleep experiments show that this product can shorten the sleep latency in mice by 35-45% and prolong sleep time by 25-35%, outperforming similar products on the market.

[0034] 5. The ternary composite structure endows the product with excellent storage stability and rehydration properties. The concentric layered structure of the organic acid calcium core, protein intermediate layer, and polysaccharide shell effectively isolates calcium ions from contact with environmental moisture and oxygen. Accelerated storage tests (40℃, 75% RH) show that after 6 months, the retention rate of γ-aminobutyric acid is ≥85% and the retention rate of isoflavone aglycones is ≥80%, which is far higher than the 60-70% retention rate of traditional soy milk powder. At the same time, the hydrophilicity of the polysaccharide shell shortens the rehydration time of the product from 3-5 minutes in the traditional process to 30-60 seconds. After rehydration, the product is evenly dispersed without precipitation, and the sensory quality is significantly improved. Attached Figure Description

[0035] Figure 1 This invention relates to the effect of germination time on γ-aminobutyric acid content and isoflavone aglycone conversion rate.

[0036] Figure 2 This invention relates to the effect of pH decrease rate on Zeta potential and calcium encapsulation efficiency.

[0037] Figure 3 This invention relates to the effect of calcium ion concentration on microcapsule particle size and calcium encapsulation efficiency.

[0038] Figure 4 The infrared Fourier transform spectrum (transmission mode) of the sample in the S1 germination and enzymatic hydrolysis stage in Example 1 of the present invention.

[0039] Figure 5 The infrared Fourier transform spectrum (transmission mode) of the sample from the S2 fermentation stage in Example 1 of this invention.

[0040] Figure 6 The infrared Fourier transform spectrum (transmission mode) of the sample in the S3 in-situ calcium nucleation stage in Example 1 of this invention.

[0041] Figure 7 The infrared Fourier transform spectrum (transmission mode) of the sample in the stage of S4 protein isoelectric point adsorption and aging to form ternary composite microcapsules in Example 1 of the present invention.

[0042] Figure 8 The infrared Fourier transform spectrum (transmission mode) of the S5 spray-dried finished soy milk powder sample in Example 1 of this invention.

[0043] Figure 9 This is a morphological diagram of the ternary in-situ composite microcapsule of Example 1 of the present invention. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0046] Example 1

[0047] A process for preparing functional soy milk powder with sleep-improving effects includes the following steps:

[0048] Example 1

[0049] The functional soy milk powder preparation process in this embodiment includes the following steps:

[0050] S1: Pretreatment and Enzymatic Digestion

[0051] Take 1000g of soybeans (protein content ≥40%, conforming to GB 1352-2009, commercially available), soak them in 5 times their volume of purified water at 22℃ for 10 hours, drain them, and place them in a germination chamber. Germinate them at 28℃ and 85% relative humidity for 30 hours, with an average sprout length of 2mm. Mix the germinated soybeans with 8 times their volume of purified water and crush them at high speed for 5 minutes to obtain germinated soybean slurry. Adjust the pH of the slurry to 7.2, raise the temperature to 52℃, and add alkaline protease Subtilisin (enzyme activity ≥200000U / g, food grade, commercially available) at a rate of 1200U / g soybean protein. Enzymatically hydrolyze the slurry in a constant temperature water bath for 60 minutes until the degree of protein hydrolysis (DH) reaches 7%. After enzymatic hydrolysis, immediately heat the slurry to 92℃ and hold for 10 minutes to inactivate the protease, then pasteurize it at 80℃ for 20 minutes. Adjust the solids content to 10% to obtain the germinated soybean hydrolysate.

[0052] S2: Fermentation and Enrichment

[0053] Cool the enzymatic hydrolysate obtained in step S1 to 35°C and inoculate it with Lactobacillus plantarum (viable count ≥10). 9 Fermentation was initiated at 35°C using a 3% (v / v) concentration of 3% (CFU / g, freeze-dried powder, commercially available) of 50% (w / v) lactic acid solution (food grade, purity ≥80%, commercially available) via a closed-loop linkage system of an online pH electrode (accuracy ±0.01pH) and a peristaltic metering pump. The pH was slowly added dropwise to decrease from 6.2 to 5.2 at a rate of 0.04pH / min. The entire fermentation process lasted 18 hours, with the pH maintained within the range of 5.15-5.25 until step S3 was performed to obtain the fermentation suspension.

[0054] S3: In-situ calcium nucleation

[0055] Add the fermentation suspension obtained in step S2 to the mixture at pH 5.2, temperature 25°C, and shear rate 150 s⁻¹. -1 Under stirring conditions, at a rate of 0.3 mL·min -1 ·L -1A calcium lactate aqueous solution (20% by mass, food grade, purity ≥98%, commercially available) was added dropwise at a rate to bring the calcium ion concentration in the system to 30 mM. The mixture was stirred for 15 min to obtain a composite dispersion containing organic acid calcium microcrystal cores.

[0056] S4: Protein Isoelectric Point Adsorption and Stabilization

[0057] Add 50% (w / v) lactic acid solution dropwise to the composite dispersion obtained in step S3 over 6 minutes to lower the pH from 5.2 to 4.7. Maintain this pH for 18 minutes to allow the fermented modified soybean protein to adsorb and coat the microcrystalline cores. Then, adjust the pH back to 5.6 using a 10% (w / v) sodium bicarbonate solution (food grade, purity ≥99.5%, commercially available), at a shear rate of 30 s⁻¹. -1 Aging for 12 minutes under certain conditions promotes the construction of a stable layer of extracellular polysaccharides on the outer layer, resulting in a ternary in-situ composite microcapsule dispersion consisting of a microcrystalline core, a protein intermediate layer, and an extracellular polysaccharide outer layer.

[0058] S5: Concentration and Spray Drying

[0059] The composite microcapsule dispersion obtained in step S4 was concentrated in a vacuum concentrator at an outlet temperature of 30°C to adjust the solid content to 36%. A spray drying tower was then used, with inlet air temperature set at 175°C, outlet air temperature at 85°C, feed temperature at 25°C, and atomization pressure at 20 MPa, to obtain a finished soy milk powder with ternary in-situ composite microcapsules as the basic unit, and a moisture content of 3.2%.

[0060] Features of Example 1: This example uses moderate parameter configurations, with all process parameters located near the midpoint of the claims scope, ensuring process stability and consistent product quality. A germination time of 30 hours and an average germination length of 2 mm fully activate glutamate decarboxylase, while a protein hydrolysis degree of 7% ensures small peptide release without excessive hydrolysis. A pH decrease rate of 0.04 pH / min and a fermentation time of 18 hours allow *Lactobacillus plantarum* to fully produce extracellular polysaccharides (polysaccharide to protein ratio approximately 2.0:1). A calcium ion concentration of 30 mM, an isoelectric point adsorption time of 18 minutes, and an aging time of 12 minutes form a stable ternary composite structure. This example is suitable for standardized production, with minimal batch-to-batch product quality fluctuations. The γ-aminobutyric acid content is approximately 220 mg / 100g, the isoflavone glycoside conversion rate is approximately 78%, the microcapsule D50 is approximately 40 μm, and the dry powder has good flowability (Carr index approximately 20), making it suitable as a basic formulation for the production of everyday functional foods.

[0061] Example 2

[0062] The functional soy milk powder preparation process in this embodiment includes the following steps:

[0063] S1: Pretreatment and Enzymatic Digestion

[0064] Take 1000g of soybeans (protein content ≥40%, conforming to GB 1352-2009, commercially available), soak them in 5 times their volume of purified water at 23℃ for 9 hours, drain them, and place them in a germination chamber. Germinate them at 27℃ and 85% relative humidity for 28 hours, with an average sprout length of 1.5mm. Mix the germinated soybeans with 8 times their volume of purified water and crush them at high speed for 5 minutes to obtain germinated soybean slurry. Adjust the pH of the slurry to 7.1, raise the temperature to 51℃, and add alkaline protease Subtilisin (enzyme activity ≥200000U / g, food grade, commercially available) at a rate of 1000U / g soybean protein. Enzymatically hydrolyze the slurry in a constant temperature water bath for 50 minutes until the degree of protein hydrolysis (DH) reaches 5%. After enzymatic hydrolysis, immediately heat the slurry to 91℃ and hold for 8 minutes to inactivate the protease, then pasteurize it at 78℃ for 18 minutes. Adjust the solids content to 9% to obtain the germinated soybean hydrolysate.

[0065] S2: Fermentation and Enrichment

[0066] Cool the enzymatic hydrolysate obtained in step S1 to 33°C and inoculate it with Lactobacillus plantarum (viable count ≥10). 9 Fermentation was initiated at 33°C with 2.5% (v / v) citric acid solution (food grade, purity ≥99.5%, commercially available) at an online pH electrode (accuracy ±0.01 pH) and a peristaltic metering pump in a closed-loop system. The pH was slowly added dropwise from 6.2 to 5.2 at a rate of 0.02 pH / min. The entire fermentation process lasted 16 hours, with the pH maintained within the range of 5.10-5.20 until step S3 was performed to obtain the fermentation suspension.

[0067] S3: In-situ calcium nucleation

[0068] Add the fermentation suspension obtained in step S2 to the mixture at pH 5.1, temperature 22°C, and shear rate 100 s⁻¹. -1 Under stirring conditions, at a rate of 0.2 mL·min -1 ·L -1 A calcium citrate aqueous suspension (15% by mass, food grade, purity ≥97%, commercially available) was added dropwise at a rate to bring the calcium ion concentration in the system to 20 mM. The mixture was stirred for 10 min to obtain a composite dispersion containing organic acid calcium microcrystal cores.

[0069] S4: Protein Isoelectric Point Adsorption and Stabilization

[0070] Add 50% (w / v) citric acid solution dropwise to the composite dispersion obtained in step S3 over 5 min to lower the pH from 5.1 to 4.65. Maintain this pH for 12 min to allow the fermented modified soybean protein to adsorb and coat the microcrystalline cores. Then, adjust the pH back to 5.5 using 10% (w / v) ammonium bicarbonate solution (food grade, purity ≥99.2%, commercially available), at a shear rate of 20 s⁻¹. -1 Aging for 8 minutes under certain conditions promotes the construction of a stable layer of extracellular polysaccharides on the outer layer, resulting in a ternary in-situ composite microcapsule dispersion consisting of a microcrystalline core, a protein intermediate layer, and an extracellular polysaccharide outer layer.

[0071] S5: Concentration and Spray Drying

[0072] The composite microcapsule dispersion obtained in step S4 was concentrated in a vacuum concentrator at an outlet temperature of 25°C to adjust the solid content to 33%. A spray drying tower was then used, with inlet air temperature set at 160°C, outlet air temperature at 78°C, feed temperature at 22°C, and atomization pressure at 18 MPa, to obtain a finished soy milk powder with ternary in-situ composite microcapsules as the basic unit and a moisture content of 2.8%.

[0073] Features of Example 2: This example employs conservative process parameters, including a slower pH decrease rate (0.02 pH / min), shorter fermentation time (16h), lower calcium ion concentration (20mM), and lower spray drying temperature (160℃ inlet air), which helps protect heat-sensitive functional components and probiotic activity. A protein hydrolysis degree of 5% moderately controls the degree of enzymatic hydrolysis, avoiding excessive hydrolysis that could affect gelation. The combination of citric acid and calcium citrate imparts a mild lemon flavor to the product, improving its mouthfeel. The lower calcium ion concentration and shorter adsorption time (12min) result in smaller microcapsule particle size (D50 approximately 30μm), contributing to a smoother texture. This example is suitable for products requiring high retention of heat-sensitive components and emphasizing probiotic survival rates, with a γ-aminobutyric acid content of approximately 180mg / 100g, an isoflavone aglycone conversion rate of approximately 73%, and a *Lactobacillus plantarum* viable count ≥10. 6 With a CFU / g content and excellent dry powder flowability (Carr index approximately 18), it is suitable as a high-end functional food or for products requiring a high live bacteria count.

[0074] Example 3

[0075] The functional soy milk powder preparation process in this embodiment includes the following steps:

[0076] S1: Pretreatment and Enzymatic Digestion

[0077] Take 1000g of soybeans (protein content ≥40%, conforming to GB 1352-2009, commercially available), soak them in 5 times their volume of purified water at 21℃ for 11 hours, drain them, and place them in a germination chamber. Germinate them at 29℃ and 85% relative humidity for 34 hours, with an average sprout length of 2.8mm. Mix the germinated soybeans with 8 times their volume of purified water and crush them at high speed for 5 minutes to obtain germinated soybean slurry. Adjust the pH of the slurry to 7.3, raise the temperature to 54℃, and add neutral protease (enzyme activity ≥150000U / g, food grade, commercially available) at 1400U / g soybean protein. Enzymatically hydrolyze the slurry in a constant temperature water bath for 70 minutes until the degree of protein hydrolysis (DH) reaches 9%. After enzymatic hydrolysis, immediately heat the slurry to 94℃ and hold for 12 minutes to inactivate the protease, then pasteurize it at 83℃ for 25 minutes. Adjust the solids content to 11% to obtain the germinated soybean hydrolysate.

[0078] S2: Fermentation and Enrichment

[0079] Cool the enzymatic hydrolysate obtained in step S1 to 36°C and inoculate it with Lactobacillus plantarum (viable count ≥10). 9 Fermentation was initiated at 36°C with 3.5% (v / v) of a lyophilized powder (CFU / g, commercially available) at a rate of ±0.01 pH. A closed-loop system using an online pH electrode (accuracy ±0.01 pH) and a peristaltic metering pump was employed to slowly add 50% (w / v) lactic acid solution (food grade, purity ≥80%, commercially available), causing the pH to decrease from 6.2 to 5.2 at a rate of 0.06 pH / min. The entire fermentation process lasted 22 hours, with the pH maintained between 5.18 and 5.28 until step S3 was performed, yielding the fermentation suspension.

[0080] S3: In-situ calcium nucleation

[0081] Add the fermentation suspension obtained in step S2 to the mixture at pH 5.3, temperature 32°C, and shear rate 250 s⁻¹. -1 Under stirring conditions, at a rate of 0.45 mL / min -1 ·L -1 A calcium malate aqueous solution (18% by mass, food grade, purity ≥96%, commercially available) was added dropwise at a rate to bring the calcium ion concentration in the system to 50 mM. The mixture was stirred for 18 min to obtain a composite dispersion containing organic calcium microcrystal cores.

[0082] S4: Protein Isoelectric Point Adsorption and Stabilization

[0083] Add 50% (w / v) lactic acid solution dropwise to the composite dispersion obtained in step S3 over 8 minutes to lower the pH from 5.3 to 4.75. Maintain this pH for 25 minutes to allow the fermented modified soybean protein to adsorb and coat the microcrystalline cores. Then, adjust the pH back to 5.7 using a 10% (w / v) sodium bicarbonate solution (food grade, purity ≥99.5%, commercially available), at a shear rate of 45 s⁻¹. -1 Aging for 18 minutes under certain conditions promotes the construction of a stable layer of extracellular polysaccharides on the outer layer, resulting in a ternary in-situ composite microcapsule dispersion consisting of a microcrystalline core, a protein intermediate layer, and an extracellular polysaccharide outer layer.

[0084] S5: Concentration and Spray Drying

[0085] The composite microcapsule dispersion obtained in step S4 was concentrated in a vacuum concentrator at an outlet temperature of 40°C to adjust the solid content to 40%. A spray drying tower was then used, with inlet air temperature set at 195°C, outlet air temperature at 95°C, feed temperature at 33°C, and atomization pressure at 24 MPa, to obtain a finished soy milk powder with ternary in-situ composite microcapsules as the basic unit and a moisture content of 3.8%.

[0086] Features of Example 3: This example employs relatively aggressive process parameters, including a longer germination time (34h), a larger average germination length (2.8mm), a higher degree of protein hydrolysis (9%), a faster pH decrease rate (0.06 pH / min), a longer fermentation time (22h), a higher calcium ion concentration (50mM), and a higher spray drying temperature (195℃ inlet air). The main optimizations are the enrichment efficiency of γ-aminobutyric acid (GABA) and isoflavone aglycones, as well as the calcium fortification effect of the microcapsules. The longer fermentation time and faster pH decrease rate result in higher extracellular polysaccharide yield (polysaccharide to protein ratio approximately 2.3:1). The high calcium concentration and longer adsorption / aging time (25min + 18min) form a denser core-shell structure, with a microcapsule D50 of approximately 65μm and a higher calcium encapsulation efficiency. This embodiment is suitable for products with high requirements for functional ingredient content and calcium fortification. It contains approximately 280 mg / 100g of γ-aminobutyric acid, about 83% isoflavone aglycone conversion rate, an organic acid calcium core-to-wall material mass ratio of approximately 0.48:1, and a total calcium content of approximately 800 mg / 100g. It is suitable as a fortified functional food or for products with high requirements for sleep improvement. Although high-temperature spraying may slightly reduce the survival rate of probiotics, the dense core-shell structure provides better protection and excellent storage stability.

[0087] Example 4

[0088] The functional soy milk powder preparation process in this embodiment includes the following steps:

[0089] S1: Pretreatment and Enzymatic Digestion

[0090] Take 1000g of soybeans (protein content ≥40%, conforming to GB 1352-2009, commercially available), soak them in 5 times their volume of purified water at 20℃ for 12 hours, drain them, and place them in a germination chamber. Germinate them at 26℃ and 85% relative humidity for 24 hours, with an average sprout length of 1mm. Mix the germinated soybeans with 8 times their volume of purified water and crush them at high speed for 5 minutes to obtain germinated soybean slurry. Adjust the pH of the slurry to 7.0, raise the temperature to 50℃, and add papain (enzyme activity ≥100000U / g, food grade, commercially available) at 800U / g soybean protein. Enzymatically hydrolyze the slurry in a constant temperature water bath for 45 minutes until the degree of protein hydrolysis (DH) reaches 4%. After enzymatic hydrolysis, immediately heat the slurry to 90℃ and hold for 5 minutes to inactivate the protease, then pasteurize it at 75℃ for 15 minutes. Adjust the solids content to 8% to obtain the germinated soybean hydrolysate.

[0091] S2: Fermentation and Enrichment

[0092] Cool the enzymatic hydrolysate obtained in step S1 to 32°C and inoculate it with Lactobacillus plantarum (viable count ≥10). 9 Fermentation was initiated at 32°C with 2% (v / v) citric acid solution (food grade, purity ≥99.5%, commercially available) added slowly via an online pH electrode (accuracy ±0.01 pH) and a peristaltic metering pump in a closed-loop system. The pH was decreased from 6.2 to 5.2 at a rate of 0.01 pH / min. The entire fermentation process lasted 12 hours, with the pH maintained between 5.05 and 5.15 until step S3 was performed to obtain the fermentation suspension.

[0093] S3: In-situ calcium nucleation

[0094] Add the fermentation suspension obtained in step S2 to the mixture at pH 5.0, temperature 20°C, and shear rate 30 s⁻¹. -1 Under stirring conditions, at a rate of 0.1 mL·min -1 ·L -1 A calcium lactate aqueous solution (20% by mass, food grade, purity ≥98%, commercially available) was added dropwise at a rate to bring the calcium ion concentration in the system to 8 mM. The mixture was stirred for 5 min to obtain a composite dispersion containing organic acid calcium microcrystal cores.

[0095] S4: Protein Isoelectric Point Adsorption and Stabilization

[0096] Add 50% (w / v) lactic acid solution dropwise to the composite dispersion obtained in step S3 over 3 minutes to lower the pH from 5.0 to 4.6. Maintain this pH for 6 minutes to allow the fermented modified soybean protein to adsorb and coat the microcrystalline cores. Then, adjust the pH back to 5.4 using a 10% (w / v) sodium bicarbonate solution (food grade, purity ≥99.5%, commercially available), and maintain this pH at a shear rate of 10 s⁻¹. -1 Aging for 5 minutes under certain conditions promotes the construction of a stable layer of extracellular polysaccharides on the outer layer, resulting in a ternary in-situ composite microcapsule dispersion consisting of a microcrystalline core, a protein intermediate layer, and an extracellular polysaccharide outer layer.

[0097] S5: Concentration and Spray Drying

[0098] The composite microcapsule dispersion obtained in step S4 was concentrated in a vacuum concentrator at an outlet temperature of 20°C to adjust the solid content to 30%. A spray drying tower was then used, with inlet air temperature set at 150°C, outlet air temperature at 75°C, feed temperature at 20°C, and atomization pressure at 15 MPa, to obtain a finished soy milk powder with a moisture content of 2.5%, using ternary in-situ composite microcapsules as the basic unit.

[0099] Example 4 Features: This example employs a boundary value verification strategy, where multiple key parameters approach or reach the boundary values ​​of the claims to verify the feasibility and process tolerance of the claims. The parameters are: germination time 24h, average sprout length 1mm, protein hydrolysis degree 4%, pH decrease rate 0.01 pH / min, fermentation time 12h, calcium ion concentration 8mM, isoelectric point adsorption time 6min, aging time 5min, solids concentration 30%, spray drying inlet air temperature 150℃, and outlet air temperature 75℃. This combination of boundary parameters demonstrates that functional soy milk powder can still be successfully prepared under relatively conservative process conditions, but the content of functional components is relatively low (γ-aminobutyric acid approximately 150mg / 100g, isoflavone glycoside conversion rate approximately 70%), microcapsule D50 approximately 5μm, and calcium content approximately 300mg / 100g. The technical rationale of this embodiment lies in the following: while the shorter germination time and lower degree of hydrolysis limit the enrichment efficiency of functional components, they preserve more of the original soybean flavor; the extremely slow pH decrease rate (0.01 pH / min), although extending the process time, provides an extremely wide operating window (approximately 100 minutes), significantly improving the controllability and batch stability for industrial scale-up; and while the low calcium concentration and short adsorption time reduce the calcium fortification effect, the microcapsule particle size is smaller, the dispersibility is better, and the taste is more delicate. This embodiment is suitable for scenarios with high requirements for process stability, emphasis on product taste delicacy, or further optimization of the basic formulation, demonstrating that even under the lower limit conditions of process parameters, the technical solution of this invention remains fully feasible.

[0100] Comparative Example 1: It is basically the same as Example 1, except that no germination treatment was performed. Soaked soybeans were directly crushed to obtain soybean slurry. The amount of other components and preparation conditions remained unchanged.

[0101] Comparative Example 2: It is basically the same as Example 1, except that the germination time is 20 hours, while the amount of other components and preparation conditions remain unchanged.

[0102] Comparative Example 3: It is basically the same as Example 1, except that the germination time is 38 hours, while the amount of other components and preparation conditions remain unchanged.

[0103] Comparative Example 4: It is basically the same as Example 1, except that the degree of protein hydrolysis (DH) is 2%, while the amount of other components and preparation conditions remain unchanged.

[0104] Comparative Example 5: Basically the same as Example 1, except that the degree of protein hydrolysis (DH) is 11%, while the amounts of other components and preparation conditions remain unchanged.

[0105] Comparative Example 6: It is basically the same as Example 1, except that in step S2, no acid is added to adjust the pH. The pH is kept constant at 6.2 throughout the fermentation process. The amount of other components and the preparation conditions remain unchanged.

[0106] Comparative Example 7: It is basically the same as Example 1, except that the fermentation time is 10 hours, while the amount of other components and preparation conditions remain unchanged.

[0107] Comparative Example 8: It is basically the same as Example 1, except that the fermentation time is 26 hours, while the amount of other components and preparation conditions remain unchanged.

[0108] Comparative Example 9: It is basically the same as Example 1, except that in-situ calcium nucleation is not performed in step S3, and the process proceeds directly to step S4. The amounts of other components and preparation conditions remain unchanged.

[0109] Comparative Example 10: Basically the same as Example 1, except that the calcium ion concentration is 70 mM, while the amounts of other components and preparation conditions remain unchanged.

[0110] Comparative Example 11: It is basically the same as Example 1, except that in step S4, the pH is not lowered to the isoelectric point for adsorption, and the pH is directly reduced from 5.2 to 5.6 for aging. The amount of other components and preparation conditions remain unchanged.

[0111] Comparative Example 12: Basically the same as Example 1, except that the isoelectric point adsorption time is 3 min, while the amount of other components and preparation conditions remain unchanged.

[0112] Comparative Example 13: It is basically the same as Example 1, except that the aging time is 3 min, while the amount of other components and preparation conditions remain unchanged.

[0113] Performance testing:

[0114] Experiment 1: Slurry Rheological Properties Test

[0115] Test Object: Ternary in-situ composite microcapsule dispersion obtained in step S4 (before concentration). Test Objective: To evaluate the rheological properties of the dispersion system under high solids content conditions and verify the technical effect of high solids content and low viscosity. Test Principle: The apparent viscosity and flow curves of the slurry at different shear rates were measured using a rheometer to characterize the flowability and stability of the dispersion system. Experimental Method: A rotational rheometer (cone-plate system, cone angle 2°, gap 50μm) was used. Dispersion samples concentrated to different solids concentrations (30%, 33%, 36%, 40%) were taken. The shear rate was measured from 1 s⁻¹ at 25℃. -1 Gradually increase to 1000 s -1 Calculate the flow and viscosity curves based on the shear stress and apparent viscosity within the specified range. Standard reference: Refer to the rheological performance test methods in GB / T 22235-2008 "Acceptance Specification for Liquid Food Packaging Equipment". Key parameters: Test temperature 25±0.5℃, shear rate range 1-1000 s⁻¹. -1 The equilibrium time at each shear rate point is 30 s. Data processing: Calculations were performed at a shear rate of 100 s for a solids concentration of 36%. -1 The apparent viscosity under the given conditions is used to evaluate the spray suitability of the slurry (a target viscosity of <500 mPa·s is preferred).

[0116] Experiment 2: Particle size and particle size distribution determination

[0117] Test Subject: Spray-dried finished soy milk powder. Test Objective: To determine the microcapsule particle size and particle size distribution, and to verify the particle size control effect of the ternary composite structure. Test Principle: The laser scattering method calculates the particle size by measuring the scattering angle and intensity distribution of laser light by the particles. Experimental Method: Using a laser particle size analyzer (Malvern Mastersizer 3000 or equivalent), 0.5g of soy milk powder sample was dispersed in 10mL of anhydrous ethanol, ultrasonically dispersed for 30s, and the measurement cell was added until the light-blocking degree was 10-15%. The particle size distribution was measured, and the measurement was repeated three times, with the average value taken. Standard Basis: Refer to GB / T 19077-2016 "Particle Size Distribution - Laser Diffraction Method". Key Parameters: Dispersant: Anhydrous ethanol; Ultrasonic power: 200W; Measurement repeatability RSD <3%; Temperature: 25℃. Data processing: Record D10, D50, D90 and span values, Span=(D90-D10) / D50, and evaluate the uniformity of particle size distribution (Span<2 indicates narrow distribution).

[0118] Experiment 3: Zeta potential measurement

[0119] Test Subject: Ternary in-situ composite microcapsule dispersion obtained in step S4 (after aging). Test Objective: To determine the Zeta potential of the microcapsule surface and evaluate the electrostatic stability of the dispersion system. Test Principle: The Zeta potential is calculated by measuring the electrophoretic velocity of charged particles in an applied electric field using electrophoretic light scattering. Experimental Method: A Zeta potential analyzer (Malvern Zetasizer Nano ZS or equivalent) was used. The dispersion sample was diluted 100 times (using deionized water, adjusting the pH to ±0.1 of the original sample pH), injected into a capillary electrophoresis tank, and the Zeta potential was measured at 25℃. Each sample was measured five times, and the average value was taken. Standard Basis: Refer to ISO 13099-2:2012 "Determination of Zeta Potential in Colloidal Systems". Key Parameters: Test temperature 25±0.1℃, dilution factor 100 times, dielectric constant 78.5 (water), viscosity 0.89 mPa·s. Data processing: Record the mean and standard deviation of the zeta potential to evaluate the dispersion stability (a dispersion system with |zeta potential|>30mV is considered stable).

[0120] Experiment 4: Powder Flowability Test

[0121] Test Subject: Spray-dried finished soy milk powder. Test Objective: To evaluate the flow properties of the dry powder and verify the effect of microcapsule structure on improving powder processability. Test Principle: The flow characteristics of the powder are characterized by measuring its loose density, tapped density, and angle of repose. Experimental Method: Using a powder comprehensive property tester, 50g of sample was accurately weighed and allowed to fall freely into a 100mL graduated cylinder through a standard funnel to determine the loose density ρ0. Then, the sample was vibrated 3000 times (amplitude 3mm, frequency 250 times / min) on a vibratory meter to determine the tapped density ρ0. t The Carr index and Hausner ratio were calculated. The angle of repose θ of the powder packing was measured using an angle of repose meter. Standards followed: GB / T 16913.3-2008 "Test Methods for Physicochemical Properties of Dust Part 3: Determination of Tapped Density" and GB / T 11986-1989 "Determination of Angle of Repose of Surfactant Powders and Particles". Key parameters: tapping amplitude 3±0.5mm, frequency 250±10 times / min, ambient temperature 20-25℃, relative humidity <60%. Data processing: The Carr index CI was calculated as follows: CI = (ρ t -ρ0) / ρ t ×100% and Hausner ratio HR=ρ t / ρ0, the liquidity evaluation criteria are: CI <15% is excellent liquidity, 15-20% is good, 21-25% is fair, and >25% is poor; the angle of repose θ <30° is excellent liquidity.

[0122] Experiment 5: Determination of γ-aminobutyric acid content

[0123] Test Subject: Finished soy milk powder. Test Objective: To quantitatively determine the content of γ-aminobutyric acid (GABA) and evaluate the synergistic enrichment effect of germination and fermentation on functional components. Test Principle: High-performance liquid chromatography (HPLC) was used to determine the GABA content through pre-column derivatization separation. Experimental Method: 2.0 g of powder sample was accurately weighed, added to 50 mL of purified water, and extracted ultrasonically for 30 min. The sample was centrifuged (4000 rpm, 10 min), and the supernatant was collected and filtered through a 0.22 μm microporous membrane. 1 mL of the filtrate was mixed with 1 mL of o-phthalaldehyde (OPA) derivatization reagent and reacted for 2 min. 10 μL of the mixture was injected for HPLC analysis. Chromatographic Conditions: C18 column (250 mm × 4.6 mm, 5 μm), mobile phase A was 0.1 mol / L sodium acetate buffer (pH 6.5), mobile phase B was methanol, gradient elution, flow rate 1.0 mL / min, column temperature 30℃, fluorescence detector (excitation wavelength 340 nm, emission wavelength 450 nm). Standard Basis: Refer to GB5009.124-2016 "National Food Safety Standard - Determination of Amino Acids in Food". Key Parameters: Extraction temperature 25℃, ultrasonic power 300W, derivatization time 2±0.2min, injection volume 10μL. Data Processing: Quantitative analysis was performed using the external standard method. A standard curve was plotted using GABA standard (purity ≥99%), with a linear range of 5-200μg / mL and R²≥0.999. The GABA content in the sample (mg / 100g) was calculated.

[0124] Experiment 6: Determination of total isoflavones and aglycone content

[0125] Test Subject: Finished soy milk powder. Test Objective: To determine the total isoflavone content and aglycone rate, and to evaluate the hydrolytic effect of β-glucosidase on isoflavone glycosides during fermentation. Test Principle: HPLC was used to determine the content of isoflavone glycosides (genistein, daidzein) and isoflavone aglycones (genistein, daidzein). Experimental Method: 1.0 g of powder sample was accurately weighed, added to 25 mL of 80% methanol solution, and extracted by reflux in an 80℃ water bath for 1 h. The supernatant was collected by centrifugation, and the extraction was repeated twice. The supernatants were combined and diluted to 50 mL, then filtered through a 0.22 μm filter membrane. HPLC conditions: C18 column (250 mm × 4.6 mm, 5 μm), mobile phase A: 0.1% glacial acetic acid aqueous solution, mobile phase B: acetonitrile, gradient elution (0-10 min 18% B, 10-30 min 18-35% B, 30-40 min 35-50% B), flow rate 1.0 mL / min, column temperature 35℃, detection wavelength 260 nm. Standard reference: GB / T 26625-2011 "Determination of Isoflavone Content in Soybeans by High Performance Liquid Chromatography in Grain and Oil Inspection". Key parameters: extraction temperature 80±2℃, reflux time 60 min, mobile phase pH 2.8-3.0. Data processing: external standard method for quantification of isoflavone components; total isoflavones = glycosides + aglycones; aglycone conversion rate = aglycone content / total isoflavones × 100%; fermentation conversion efficiency was evaluated (target aglycone conversion rate ≥ 75%).

[0126] Experiment 7: Live count of Lactobacillus plantarum

[0127] Test Subject: Finished soy milk powder. Test Objective: To determine the viable count of *Lactobacillus plantarum* in the product and evaluate the protective effect of spray drying on probiotics. Test Principle: The plate count method isolates and counts target strains using selective culture media. Experimental Method: Under aseptic conditions, weigh 10g of the powder sample, add 90mL of sterile physiological saline (0.85% NaCl), homogenize in a beater for 2min to prepare a 1:10 dilution, and serially dilute 10-fold with sterile physiological saline to a final concentration of 10. -5 -10 -71 mL of the diluted solution was poured onto MRS agar medium (Bengali red agar, used for selective culture of lactic acid bacteria). Two replicates were performed for each dilution. The culture was carried out at 37℃ under anaerobic conditions (anaerobic tank or anaerobic incubator, 5% CO2, 10% H2, 85% N2) for 48-72 hours, and colonies were counted. Standard reference: GB 4789.35-2016 "National Food Safety Standard - Microbiological Examination of Food - Examination of Lactic Acid Bacteria". Key parameters: culture temperature 37±1℃, culture time 48-72 hours, anaerobic environment (oxygen concentration <1%), dilution preparation time <15 minutes. Data processing: Plates with colony counts between 30-300 were used for counting. The viable count (CFU / g) was calculated as: average colony count × dilution factor × 10. Each sample was measured three times, and the average value was taken to evaluate the probiotic survival effect (target ≥10). 6 CFU / g).

[0128] Experiment 8: Determination of calcium encapsulation efficiency in microcapsules

[0129] Test Subject: Finished soy milk powder. Test Objective: To determine the encapsulation efficiency of microcapsules for the organic acid calcium core and verify the stability of the ternary composite structure. Test Principle: The encapsulation efficiency is calculated by measuring the surface free calcium and total calcium content. Experimental Method: Surface Free Calcium Determination: Take 1.0g of powder sample, add 25mL of deionized water, shake at 25℃ for 30min (simulating surface calcium dissolution), centrifuge (4000rpm, 10min), collect the supernatant, and determine the calcium content using atomic absorption spectrometry (AAS) or inductively coupled plasma optical emission spectrometry (ICP-OES), recorded as C1. Total Calcium Determination: Take 1.0g of powder sample, ashing at 450℃ for 4h, dissolve the ash in 10mL of 6mol / L HCl, and bring the volume to 50mL. Determine the total calcium content using AAS or ICP-OES, recorded as C2. Standard Basis: Refer to GB5009.92-2016 "National Food Safety Standard - Determination of Calcium in Food". Key parameters: oscillation temperature 25±2℃, oscillation frequency 150rpm, ashing temperature 450±10℃. Data processing: Encapsulation efficiency (%) = (C2-C1) / C2×100%, each sample was measured 3 times and the average value was taken to evaluate the microcapsule encapsulation effect (target encapsulation efficiency ≥85%). To verify the scientific validity and feasibility of the technical solution of this invention, based on fixed soybean dosage of 1000g, soaking conditions (temperature 22℃, time 10h), germination environment (temperature 28℃, humidity 85%), enzymatic hydrolysis parameters (temperature 52℃, enzyme addition of 1200U / g protein, time 60min, degree of hydrolysis 7%), fermentation conditions (temperature 35℃, inoculum size 3%, pH decrease rate 0.04 pH / min, time 18h), calcium concentration 30mM, isoelectric point adsorption time 18min, aging time 12min, solids concentration 36%, and spray drying process (inlet air temperature 175℃, outlet air temperature 85℃), a systematic single-factor investigation was conducted to reveal the influence of key process parameters on the functional characteristics of the product. Figure 1 The verification results showed that when the germination time was in the range of 30-34h, the γ-aminobutyric acid content reached the optimal range of 245-255mg / 100g and the isoflavone glycoside conversion rate reached 78-82%. When the germination time was too short (<24h), the activation of glutamate decarboxylase was insufficient, resulting in low GABA enrichment efficiency. When the germination time was too long (>36h), the excessive consumption of the generated GABA by soybean germination for amino acid metabolism caused the content to decrease. This precise definition of the germination time window confirms the key role of germination pretreatment in the enrichment of functional components and verifies the scientific basis for selecting a 32h germination time in this invention. Figure 2The verification data showed that when the pH decrease rate was 0.04-0.06 pH / min, the absolute value of the Zeta potential on the microcapsule surface and the calcium encapsulation efficiency reached the optimal levels of 38-39 mV and 88-89%, respectively. When the pH decrease rate was too slow (<0.02 pH / min), the fermentation time was prolonged, but the extracellular polysaccharide production was insufficient, resulting in insufficient thickness of the stabilizing layer and affecting the encapsulation effect. When the pH decrease rate was too fast (>0.08 pH / min), Lactobacillus plantarum did not have enough time to produce extracellular polysaccharides, and the drastic pH fluctuations damaged the integrity of the microcapsule structure. The revelation of this pH gradient regulation law proves the necessity of dynamic pH control for constructing a stable ternary core-shell structure and verifies the rationality of the 0.05 pH / min decrease rate used in this invention. Figure 3 The verification results show that when the calcium ion concentration is 30-50 mM, the microcapsule particle size and encapsulation efficiency are within the ideal range. The particle size of 40-68 μm is suitable for spray drying process, and the encapsulation efficiency of 88-92% ensures the calcium strengthening effect. When the calcium concentration is too low (<15 mM), the number of nuclei is small, resulting in small particle size but insufficient encapsulation efficiency. When the calcium concentration is too high (>60 mM), the nucleation density is too large, resulting in increased microcapsule particle size and easy aggregation. Although the encapsulation efficiency is slightly higher, the flowability is worse. This quantitative relationship between calcium concentration and microcapsule structure and performance confirms the importance of calcium concentration balance regulation for microcapsule quality control and verifies the scientific nature of the 40 mM calcium concentration selected in this invention. Based on the results of the three verification experiments above, the technical approach of this invention—achieving efficient enrichment of functional components through precise control of germination time, constructing a stable core-shell structure through dynamic pH gradient regulation, and achieving synergistic improvement in particle size and encapsulation efficiency through calcium concentration balance optimization—has been fully verified. This demonstrates that the process is theoretically sound, operationally reliable and controllable, and significantly effective, providing solid experimental evidence and data support for the industrial application of this invention.

[0130] Figure 4 The transmission FTIR spectra of the S1 germination and enzymatic digestion samples are shown. The main protein bands Amide I / II are located at approximately 1652 / 1544 cm⁻¹. -1 Accompanied by a weak 1730 cm -1 carbonyl group, 3300 cm -1 The nearby broadband corresponds to OH / NH hydrogen bonds, 1080-1045 cm⁻¹ -1 Moderate C–O signaling suggests the formation of small sugars / peptides, but the intensity is limited; Figure 5 It exhibits the S2 fermentation stage, with depths of 1153, 1080, and 1045 cm. -1 Polysaccharide-related COC / CO bands were significantly enhanced and reached 1730 cm⁻¹. -1 Free lactic acid is still identifiable; Amide I / II is approximately 1650 / 1542 cm⁻¹. -1Band-shaped fine-tuning indicates the establishment of weak interactions between proteins and organic acids / polysaccharides; Figure 6 This represents the S3 in-situ calcium nucleation stage, with lactate COO-ν_as / ν_s at approximately 1578 / 1413 cm⁻¹. -1 Appears in pairs and is enhanced, and 1730 cm -1 Further weakening confirms Ca 2+ It forms salts with lactate and creates organic acid calcium microcrystal cores, while the micro-displacement and narrowing of the Amide bands reflect changes in the protein microenvironment. Figure 7 Following adsorption and aging at the corresponding S4 isoelectric point, ternary composite microcapsules are formed. Amide I, with a thickness of 1650 cm⁻¹, forms these microcapsules. -1 Migrating to 1628 cm -1 It also enhances the indication of β-sheet / aggregate state rise and shell compaction, COO - Paired peaks are maintained at 1153, 1070, and 1042 cm⁻¹. -1 The polysaccharide bands were further enhanced, indicating that the outer EPS layer was stably constructed; Figure 8 For the S5 spray-dried product, the OH broadband and baseline convergence indicate a decrease in moisture content and dry densification, with Amide I / II stabilizing at 1634 / 1534 cm⁻¹. -1 And 1578 / 1413 cm -1 COO - The paired peaks and the polysaccharide fingerprint region are preserved, indicating that drying did not damage the composite structure; Figure 9 The morphology of the ternary in-situ composite microcapsules prepared in Example 1 of this invention was observed by scanning electron microscopy. The microcapsules exhibited a regular spherical or near-spherical structure with a smooth, dense surface free of obvious cracks and pores. A continuous, stable layer formed by extracellular polysaccharides tightly enveloped the protein adsorption layer on the surface of the microcapsules. Local magnified images showed a distinct core-shell interface, exhibiting typical ternary composite structural characteristics. This complete and dense microcapsule morphology confirmed the effectiveness of the three-step construction process of in-situ calcium nucleation, isoelectric point adsorption, and extracellular polysaccharide stabilization. It verified the feasibility of the technical route of achieving hydrophobic adsorption of proteins near their isoelectric point and electrostatic adsorption of extracellular polysaccharides to form a core-shell structure layer by layer. This demonstrates that the ternary in-situ composite microcapsules constructed in this invention possess excellent qualities of structural integrity, clear layers, and uniform dispersion, laying a solid microstructural foundation for the functional stability and industrial application of the product.

[0131] The performance of the soy milk powders from the examples and comparative examples is summarized in Table 1. Comparative Example 1, due to the lack of germination treatment, did not activate glutamate decarboxylase in the soybeans, resulting in extremely low GABA enrichment efficiency. Furthermore, the lack of preliminary protein modification during germination led to insufficient subsequent enzymatic hydrolysis, significantly reducing isoflavone aglycone conversion rate. Incomplete microcapsule structure resulted in high slurry viscosity and poor flowability. Comparative Examples 2 and 3, due to excessively short or long germination times respectively, showed different outcomes. The former failed to fully activate glutamate decarboxylase and had insufficient protein pre-modification, while the latter, due to excessive germination, caused soybean germination to consume the already generated GABA for amino acid metabolism. Meanwhile, excessively long germination time leads to excessively large germination bodies, increasing the difficulty of subsequent crushing and affecting particle size uniformity. Both deviate from the optimal germination window, resulting in a decrease in the enrichment efficiency of functional components. Comparative Examples 4 and 5 have excessively low or high protein hydrolysis. The former has insufficient small peptide substrates, which limits the metabolic efficiency of Lactobacillus plantarum during fermentation, inhibiting both GABA production and isoflavone conversion. The latter has excessive hydrolysis, which destroys the emulsification and gelation properties of proteins, leading to decreased microcapsule structural stability, abnormally high slurry viscosity, and reduced viable cell count. Comparative Example 6 lacks dynamic pH gradient control, maintaining pH 6 throughout the fermentation process.2. This resulted in limited acid production and insufficient extracellular polysaccharide synthesis in *Lactobacillus plantarum*. Simultaneously, high pH inhibited β-glucosidase activity, significantly reducing isoflavone aglycone conversion. Although the viable count of probiotics was high under suitable pH conditions, the enrichment of functional components was poor. Furthermore, the lack of pH-induced protein conformational changes led to decreased microcapsule dispersion stability, a significant increase in slurry viscosity, and a decrease in the absolute value of the zeta potential. Comparative Examples 7 and 8, due to excessively short or long fermentation times, respectively, resulted in insufficient extracellular polysaccharide production, incomplete GABA accumulation, and inadequate isoflavone conversion. The latter, although… While the high content of functional components led to excessive fermentation, causing *Lactobacillus plantarum* to enter its death phase and resulting in a decrease in viable cell count, prolonged low pH environments could cause excessive protein aggregation, affecting microcapsule stability. Comparative Example 9 skipped the in-situ calcium nucleation step, lacking an organic acid calcium core, causing the microcapsules to lose their core-shell structure. Although the particle size was small, the calcium encapsulation rate was extremely low, failing to achieve calcium fortification. Furthermore, the lack of a calcium core as a nucleation center led to the formation of irregular aggregates of the protein-polysaccharide complex, affecting dispersion uniformity. Comparative Example 10, due to excessively high calcium ion concentration, nucleation… Excessive density leads to a significant increase in microcapsule particle size, making aggregation more likely. Although the calcium encapsulation efficiency is slightly improved, the excessively large particle size severely deteriorates powder flowability. At the same time, high calcium concentration may cause excessive cross-linking with proteins, resulting in a significant increase in slurry viscosity and absolute value of Zeta potential, but the dispersion stability decreases. In Comparative Example 11, due to the omission of the isoelectric point adsorption step, the protein failed to form a dense intermediate coating layer near the isoelectric point through hydrophobic interactions and hydrogen bonds, resulting in exposed calcium core and extremely low calcium encapsulation efficiency. Furthermore, the lack of buffering effect of the protein layer allows the outer layer of extracellular polysaccharide to directly contact the calcium core, leading to structural instability. Although skipping the adsorption step reduces the particle size, the integrity of the microcapsules is damaged. Comparative Examples 12 and 13 suffer from insufficient isoelectric point adsorption time or aging time, respectively. The former results in insufficient adsorption of proteins on the calcium core surface, insufficient coating layer thickness and density, while the latter results in insufficient aging time, leading to insufficient stabilization of the protein-polysaccharide complex and incomplete establishment of the hydrogen bond network. Both lead to loose microcapsule structure, decreased calcium encapsulation efficiency, and the incomplete core-shell structure is easily damaged during spray drying, affecting the quality of the final product. .

[0132] Table 1. Data on Functional Components and Calcium Nutritional Properties

[0133] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that any equivalent structural transformations made under the concept of the present invention and using the contents of the specification and drawings of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A preparation process for functional soy milk powder with sleep-improving effects, characterized in that, Includes the following steps: S1: Pretreatment and enzymatic hydrolysis: Soak soybeans and then germinate them to obtain germinated soybean enzymatic hydrolysate; heat the hydrolysate to 90-95℃ and hold for 5-15 minutes to inactivate the protease; then pasteurize at 75-85℃ for 15-30 minutes. S2: Fermentation and enrichment: After cooling the enzymatic hydrolysate obtained in step S1 to 32-37℃, inoculate it with Lactobacillus plantarum for fermentation at an inoculation amount of 2-4% (v / v). Ferment at 32-37℃ for 12-24 h. Add lactic acid or citric acid online to decrease the pH from 6.2 to 5.2 at a rate of 0.01-0.08 pH / min and maintain it until step S3 is performed to obtain a fermentation suspension. S3: In-situ calcium nucleation: Add an organic acid calcium solution dropwise to the fermentation suspension obtained in step S2 to generate organic acid calcium microcrystal nuclei in situ within the system, thus obtaining a composite dispersion containing organic acid calcium microcrystal nuclei; S4: Protein isoelectric point adsorption and stabilization: Adjust the pH of the composite dispersion obtained in step S3 to near the isoelectric point so that fermented modified soybean protein adsorbs and coats the microcrystal core. Then adjust the pH and age it to promote the construction of a stable layer of self-generated extracellular polysaccharide in the same fermentation system on the outer layer, and obtain a ternary in-situ composite microcapsule dispersion with microcrystal core, protein intermediate layer and extracellular polysaccharide outer layer. S5: Concentration and spray drying: The composite microcapsule dispersion obtained in step S4 is concentrated and then spray dried to obtain the finished soy milk powder with ternary in-situ composite microcapsules as the basic unit.

2. The preparation process of a functional soy milk powder with sleep-improving effect as described in claim 1, characterized in that, In step S1, soybeans are soaked at 20-25℃ for 8-12 hours and germinated at 26-30℃ for 24-36 hours, with an average sprout length of 1-3 mm.

3. The preparation process of a functional soy milk powder with sleep-improving effect as described in claim 1, characterized in that, In step S1, alkaline protease, neutral protease, or papain are added at pH 7.0-7.4 and temperature 50-55℃ for 45-75 min to hydrolyze the protein to a degree of hydrolysis (DH) of 4-10%, and the solids mass fraction in the solution is adjusted to 8-12%.

4. The preparation process of a functional soy milk powder with sleep-improving effect as described in claim 1, characterized in that, The alkaline protease is Subtilisin.

5. The preparation process of a functional soy milk powder with sleep-improving effect as described in claim 1, characterized in that, In step S2, lactic acid or citric acid is added online to decrease the pH from 6.2 to 5.2 at a rate of 0.01-0.08 pH / min and maintain this pH until step S3 is performed.

6. The preparation process of a functional soy milk powder with sleep-improving effect as described in claim 1, characterized in that, The pH control employs a closed-loop linkage between an online pH electrode and a metering pump, with a pH decrease rate of 0.03-0.05 pH / min and an endpoint pH value of 5.1-5.

3.

7. The preparation process of a functional soy milk powder with sleep-improving effect as described in claim 1, characterized in that, In step S3, under conditions of pH 5.0-5.4 and a system shear rate of 30-300 s⁻¹,... -1 Add an aqueous solution or aqueous suspension of calcium lactate, calcium citrate, or calcium malate dropwise at a rate of 0.1-0.5 mL / min. -1 ·L -1 The calcium ion concentration in the system is 8-60 mM and maintained for 5-20 min, while the system temperature is maintained at 20-35℃ during the dropwise addition.

8. The preparation process of a functional soy milk powder with sleep-improving effect as described in claim 1, characterized in that, In step S4, the pH of the composite dispersion is decreased from 5.0-5.4 to 4.6-4.8 within 3-10 min and maintained for 6-30 min. Subsequently, the pH is adjusted back to 5.4-5.8 using sodium bicarbonate or ammonium bicarbonate at a shear rate of 10-50 s. -1 Aging under these conditions for 5-20 minutes.

9. The preparation process of a functional soy milk powder with sleep-improving effect as described in claim 5, characterized in that, In step S5, concentration is carried out at an outlet temperature of 20-45℃ to adjust the solid mass fraction in the concentrated liquid to 30-42%; spray drying is carried out at an inlet air temperature of 150-200℃, an outlet air temperature of 75-100℃, and an inlet temperature of 20-35℃.

10. A functional soy milk powder with sleep-improving effects prepared by the preparation method according to any one of claims 1-9, characterized in that, The soy milk powder includes ternary in-situ composite microcapsules, which consist of an organic acid calcium microcrystal core, a fermented modified soybean protein intermediate layer, and a self-generated extracellular polysaccharide shell from Lactobacillus plantarum derived from the same fermentation system. The wall material is composed of extracellular polysaccharides generated by the fermentation system and fermentation-modified soybean protein, without the need to add exogenous wall materials; The microcapsules have a D50 of 5-80 μm; The soy milk powder contains 150-300 mg / 100 g of γ-aminobutyric acid and has an isoflavone aglycone conversion rate of 70-85%. The mass ratio of the extracellular polysaccharide to the fermented modified soybean protein is 1.5-2.5:1 on a dry basis, and the mass ratio of the organic acid calcium core to the wall material is 0.35-0.50:1.

Images