Special enzyme composition for soybean milk powder for infants and preparation method of special enzyme composition

Abstract

The invention belongs to the technical field of food, and provides a special enzyme composition for infant soybean milk powder and soybean milk powder thereof. A three-stage directional enzymolysis technology is adopted, the three-stage directional enzymolysis technology comprises glutamine transaminase deamidation pretreatment, bacillus subtilis protease-trypsin-papain first-stage hydrolysis and aminopeptidase-phytase-beta-glucosidase second-stage hydrolysis, and conductivity control, carbon dioxide ventilation in-situ complexation and spray drying processes are combined. The free amino nitrogen of the product is increased by more than or equal to 20%, the proportion of 200-1500Da peptide fragments is more than or equal to 70%, the median particle size is 50-180 microns, the water activity is less than or equal to 0.30, the instant dissolving time is less than or equal to 30 seconds at 25 DEG C under the water hardness of 15-200ppm, the settling rate is less than or equal to 3% after standing for 30 minutes, and the zeta potential is-10 to-25mV. The palatability contradiction between deep hydrolysis and bitterness inhibition of the bean-based infant formula powder, the brewing contradiction between high-calcium reinforcement and instant stability and the process controllability contradiction between three-stage enzymolysis and ionic strength accumulation are effectively solved, and the bean-based infant formula powder has wide application value in the fields of bean-based infant formula food and vegetable protein nutritional products.

Description

TECHNICAL FIELD

[0001] The application relates to the field of food technology, in particular to an enzyme composition special for infant soy milk powder and soy milk powder. BACKGROUND

[0002] With the increasing incidence of infant milk protein allergy and the rapid development of plant-based food consumption trends, soy-based infant formula powder as a substitute for cow milk protein is increasingly valued in the global market. The digestive system of infants and young children has not yet fully developed, and their ability to digest and absorb protein is limited, so soy-based formula powder needs to be degraded into small molecular peptide segments that are easy to digest and absorb through deep hydrolysis technology, while maintaining a suitable balance of amino acids to meet the needs of growth and development. In addition, infant formula powder has a high requirement for the fortification level of minerals such as calcium, and the calcium content usually needs to reach 400-800 mg / 100 g to support bone development, which requires the product to maintain good solubility and stability under high calcium addition conditions to avoid quality problems such as precipitation, caking and other factors that affect feeding experience. At the same time, bitter peptides are easily produced during the deep hydrolysis of soy-based protein, which affects the palatability of the product, and how to effectively inhibit bitterness while achieving sufficient hydrolysis is a key challenge in the development of soy-based infant formula powder. At the industrial production level, the three-stage enzymatic hydrolysis process requires precise control of parameters such as pH, temperature and time in each stage, and the accumulation of ionic strength caused by the release of salts during enzymatic hydrolysis can interfere with the efficiency of subsequent enzymatic reactions and the functional properties of the product, so establishing an ionic strength-controllable enzymatic hydrolysis system is of great significance to ensure the stability of product quality and process reproducibility. The degree of satisfaction of the above performance requirements directly determines whether the soy-based infant formula powder can reach the level of cow milk-based formula powder in terms of nutritional value, sensory quality, and convenience of preparation, thereby expanding the application range of the product and promoting the progress of the industry.

[0003] Although soy protein deep hydrolysis technology has been widely used in the food industry, there are still significant deficiencies in the development of soy-based infant formula powder. For example, Chinese Patent No. CN106987612A discloses a soy protein enzymatic product and its preparation method and application, but there are problems of insufficient hydrolysis caused by single protease type and high content of bitter peptides, because the single enzyme system has limited action sites on the protein substrate, cannot achieve multi-point cutting and precise control of the peptide chain, and cannot deamidate glutamine residues in soy protein to reduce bitter precursors. Chinese Patent No. CN103243079B discloses an extraction method of duck chondroitin sulfate complex enzymolysis, but there are problems of decreased instant solubility and poor storage stability after chelation of the enzymatic product with calcium ions, because the technology does not establish an in-situ complexation mechanism, but uses a post-added calcium salt method, resulting in the formation of large particle precipitates of calcium salt and negatively charged peptides in the solution, and lack of precise control of microstructure parameters such as powder particle size distribution and zeta potential. Chinese Patent No. CN111758967B discloses a giant salamander collagen peptide composition and its application, but there are problems of complex process control and ion strength accumulation leading to decreased enzyme activity, because salt by-products accumulate continuously in the multi-stage enzymolysis process, the system conductivity increases to inhibit the efficiency of enzyme-catalyzed reactions, and no intermediate conductivity control step is set, and the reaction conditions of each enzyme are not optimized for orderly separation and synergistic effect. In addition, the existing technology lacks systematic design of key parameters such as the mass ratio of enzymes in the enzyme composition, the matching of reaction conditions, and the use sequence, resulting in a wide range of peptide molecular weight distribution, low content of functional peptides, and insufficient increase in free amino nitrogen, which makes it difficult to meet the multiple requirements of infant digestion and absorption, nutritional density, sensory quality, and stable industrial production. SUMMARY

[0004] The purpose of the present application is to provide an enzyme composition for infant soy milk powder and a preparation method thereof, to solve the problems of the current soy-based infant formula powder in the contradiction between deep directional hydrolysis and bitter taste inhibition, the contradiction between high calcium fortification and instant solubility stability, and the process controllability contradiction between three-stage enzymolysis precise control and ion strength accumulation, and to realize the multi-objective synergistic optimization of infant digestion and absorption, nutritional density, sensory quality, and stable industrial production.

[0005] This invention constructs a three-stage directional enzymatic hydrolysis system, which mainly involves deamidation-based TGase pretreatment, limiting cross-linking, and first-stage hydrolysis by Bacillus subtilis protease-trypsin-papain, followed by second-stage hydrolysis by aminopeptidase-phytase-β-glucosidase. Combined with innovative process units such as water replenishment or ultrafiltration conductivity regulation, in-situ complexation with carbon dioxide ventilation, and instantaneous melting treatment on the surface of spray-dried powders, this invention achieves a triple synergistic effect: optimized palatability through deep protein hydrolysis and bitterness inhibition, improved reconstituted quality through high calcium chelation and rapid dissolution stability, and enhanced process controllability through multi-stage enzymatic hydrolysis synergy and ionic strength control. This synergistic effect is manifested in the following aspects: the deamidation pretreatment reduces the content of glutamine residues, a bitter precursor, creating a low-bitter substrate basis for subsequent enzymatic hydrolysis; the two-stage differentiated hydrolysis precisely regulates the molecular weight distribution of peptides, ensuring that the proportion of functional peptides with a molecular weight of 200-1500 Da is not less than 70%; the conductivity regulation eliminates the inhibition of enzyme activity by salt accumulation, ensuring the efficient progress of each stage of enzymatic reaction; and the in-situ complexation places the chelation process of calcium ions with negatively charged peptides before spray drying, avoiding precipitation and clumping problems caused by the subsequent addition of calcium salts, ultimately achieving a multi-dimensional synergistic improvement in nutrition, function, sensory qualities, and processing quality.

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

[0007] An enzyme composition for infant soy milk powder, comprising, by weight, 2-8 parts of transglutaminase, 25-35 parts of Bacillus subtilis protease, 20-30 parts of trypsin, 10-20 parts of papain, 10-15 parts of aminopeptidase, 5-10 parts of phytase, and 1-5 parts of β-glucosidase; wherein the aminopeptidase is derived from Aspergillus oryzae.

[0008] Furthermore, the reaction pH of the transglutaminase is 6.0-6.8, and the reaction temperature is 35-45℃; the reaction pH of the Bacillus subtilis protease, trypsin, and papain is 7.6-8.0, and the reaction temperature is 50-55℃; the reaction pH of the aminopeptidase, phytase, and β-glucosidase is 7.0-7.2, and the reaction temperature is 52-55℃.

[0009] Furthermore, the order of use of the enzyme composition is as follows: first, the transglutaminase is added for deamidation pretreatment; then, the Bacillus subtilis protease, trypsin, and papain are added for the first stage of hydrolysis; and then the aminopeptidase, phytase, and β-glucosidase are added for the second stage of hydrolysis. The reaction time for the deamidation pretreatment is 60-90 min; the reaction time for the first stage of hydrolysis is 60-90 min; and the reaction time for the second stage of hydrolysis is 30-60 min. The free amino nitrogen after deamidation pretreatment is increased by no less than 20%.

[0010] Furthermore, in the enzyme composition, the mass ratio of Bacillus subtilis protease, trypsin and papain is 25-35:20-30:10-20.

[0011] Furthermore, the enzyme composition further comprises α-galactosidase, and its mass fraction in the composition is 1-5 parts; the reaction conditions of the α-galactosidase are: pH 4.8-5.2, temperature 45-55℃, time 20-40 min; so that the total amount of raffinose and stachyose is reduced by not less than 80%.

[0012] Furthermore, in the enzyme composition, the transglutaminase has an enzyme activity of 100-500 U / g, and the Bacillus subtilis protease has an enzyme activity of 50,000-200,000 U / g.

[0013] As a concept of this invention, the directional hydrolysis design employing seven enzymes in synergy is primarily used to enhance the digestibility, palatability, and nutritional functionality of soy-based infant formula. Transglutaminase, as a deamidation pretreatment enzyme, converts glutamine residues in soy protein into glutamate, reducing the precursors for bitter peptides generated during subsequent hydrolysis. Simultaneously, it increases the surface hydrophobicity and charge density of the protein, enhancing the enzymatic hydrolysis efficiency of subsequent proteases. Bacillus subtilis protease, trypsin, and papain, as the main enzymes in the first stage of hydrolysis, possess differentiated substrate specificity and cleavage sites. Bacillus subtilis protease exhibits high activity towards peptide bonds following basic amino acid residues; trypsin specifically cleaves the carboxyl-terminal peptide bonds of lysine and arginine; and papain has broad-spectrum activity towards peptide bonds following hydrophobic amino acid residues. The synergistic effect of these three enzymes achieves multi-point cleavage and initial hydrolysis of soy protein, degrading large protein molecules into medium-molecular-weight peptides, laying the foundation for the second stage of refined hydrolysis. Aminopeptidase, derived from Aspergillus oryzae, possesses the ability to continuously cleave N-terminal amino acids in peptide chains. It can further degrade the medium-sized peptides produced in the first stage of hydrolysis into small peptides and free amino acids, significantly increasing the content of free amino nitrogen and enhancing the product's solubility and nutrient absorption efficiency. Phytase degrades phytic acid in soybeans, releasing chelated minerals such as calcium, iron, and zinc, improving mineral bioavailability, and simultaneously eliminating the inhibitory effect of phytic acid on protein digestion and absorption. β-glucosidase hydrolyzes glycosides such as daidzein in soybeans, reducing the content of anti-nutritional factors and improving sensory quality. These seven enzymes work in an orderly manner under different reaction conditions, employing a three-stage progressive hydrolysis strategy—deamidation pretreatment, multi-point cleavage hydrolysis in the first stage, and refined degradation in the second stage—to precisely control the molecular weight distribution of peptides. This ensures that the mass fraction of functional peptides (200-1500 Da) is not less than 70%, while controlling the increase in free amino nitrogen to not less than 20%, achieving a synergistic effect of deep hydrolysis, bitterness inhibition, nutrient fortification, and functional peptide enrichment.

[0014] A type of soy milk powder is prepared by enzymatic hydrolysis of soy protein isolate using the above-mentioned enzyme composition specifically for infant soy milk powder, followed by in-situ complexation and spray drying. The median particle size (D50) of the soy milk powder is 50-180 μm, and the water activity is not higher than 0.30. When prepared at a temperature of 25℃ and a water hardness of 15-200 ppm, the instant dissolution time is not higher than 30 s, and the sedimentation rate after standing for 30 min is not higher than 3%.

[0015] Furthermore, the zeta potential is -10 to -25 mV, and the median particle diameter drift is less than 15% when stored at 40℃ and 75% relative humidity for 4-8 weeks. The mass fraction of peptides with molecular weights of 200-1500 Da is not less than 70%.

[0016] Furthermore, the soy milk powder has a protein content of 15-25 g / 100 g and a calcium content of 400-800 mg / 100 g.

[0017] The preparation method of soy milk powder includes the following steps: S1 Using soy protein isolate as raw material, deamidation is performed using transglutaminase; S2 Using a special enzyme composition for infant soy milk powder, two-stage directional hydrolysis is performed, with Bacillus subtilis protease, trypsin, and papain added in the first stage, and aminopeptidase, phytase, and β-glucosidase added in the second stage; S3 Between the two stages of S2, the conductivity of the system is controlled to be no higher than 2.5 mS / cm by adding water or ultrafiltration; S4 Carbon dioxide is introduced, and in-situ complexation is performed using calcium carbonate as a calcium source; S5 Citric acid is added; S6 A terminal heat inactivation and sterilization step is set; S7 Spray drying is performed.

[0018] Furthermore, the reaction conditions for transglutaminase in S1 are: pH 6.0-6.8, temperature 35-45℃, time 60-90 min, and dosage of 1000-8000 U / kg base protein; the reaction conditions for the first stage in S2 are: pH 7.6-8.0, temperature 50-55℃, time 60-90 min, and total enzyme activity dosage of 5000-20000 U / kg base protein; the reaction conditions for the second stage are: pH 7.0-7.2, temperature 52-55℃, time 30-60 min, and total enzyme activity dosage of 2000-10000 U / kg base protein.

[0019] Furthermore, the reaction conditions in S4 are: pH 5.4-5.8, temperature 40-55℃, molar ratio of peptide carboxyl groups to calcium ions 3-8:1, and ripening time 10-20 min; in S5, the molar ratio of citric acid to calcium is 0.1-0.3, the pH is maintained at 5.4-5.8, and the ripening time is 10-30 min; in S6, the temperature is heated to 75-85℃ and maintained for 5-15 min; in S7, the outlet temperature is 65-70℃, and instantaneous melting treatment of the powder surface is performed.

[0020] Furthermore, in the preparation method described above, the enzyme composition is used as a processing aid, and the residual enzyme activity in the final product is not higher than 5%.

[0021] As another aspect of this invention, the integrated preparation design of enzymatic hydrolysis-in-situ complexation-spray drying is mainly used to enhance the instant solubility, storage stability, and reconstitution quality of soy milk powder. Using soy protein isolate as raw material, a hydrolysate rich in functional peptides is obtained through three-stage directional enzymatic hydrolysis, wherein peptides with a molecular weight range of 200-1500 Da account for no less than 70%. Peptides in this molecular weight range have good solubility, surface activity, and bioactivity, effectively promoting the digestion and absorption of protein by infants and young children. In-situ complexation technology is one of the key innovations of this invention. By introducing carbon dioxide into the hydrolysate after enzymatic hydrolysis, the pH value of the system is reduced to a suitable range of 5.4-5.8. At this time, calcium carbonate is added as a calcium source. The calcium carbonate gradually dissolves in the carbon dioxide-water system, releasing calcium ions. The calcium ions and the negatively charged peptide carboxyl groups form a stable calcium-peptide chelate through electrostatic interaction and coordination bonds. This chelation process is carried out in-situ in the liquid phase, avoiding the formation of large particle precipitates caused by the subsequent addition of calcium salts. At the same time, by controlling the molar ratio of peptide carboxyl groups to calcium ions to 3-8:1, the stability and solubility of the chelate are ensured. Citric acid is added as an auxiliary chelating agent, with the molar ratio of citric acid to calcium controlled at 0.1-0.3. Citric acid can form soluble chelates with calcium ions and also form hydrogen bond networks with peptides through its carboxyl groups, further enhancing the stability of the chelation system. At the same time, it maintains a slightly acidic pH range (pH 5.4–5.8) above the isoelectric point (after deamidation / hydrolysis), preventing the product from undergoing further enzymatic reactions during storage that could lead to quality deterioration. It also achieves terminal heat inactivation and sterilization to ensure commercial sterility requirements and guarantee product safety.

[0022] The mechanism of action and synergistic effect of the complex protease system of transglutaminase and Bacillus subtilis protease-trypsin-papain in the preparation of soy-based infant formula powder in this invention are reflected in the following aspects. The main function of transglutaminase is to pre-treat soy protein by deamidation. It catalyzes the deamination reaction of the γ-amide group of glutamine residues in protein molecules to generate glutamate. This process increases the negative charge density of protein molecules, lowers the isoelectric point, and enhances surface hydrophobicity, thereby improving the solubility, emulsification, and foaming properties of proteins. More importantly, the deamidation treatment reduces the formation of bitter peptide precursors during subsequent hydrolysis. This is because untreated glutamine residues are prone to generating bitter glutamine peptides under the action of proteases. The deamidation pre-treatment reduces the formation of bitter substances from the source. Bacillus subtilis protease, trypsin, and papain, as a complex protease system for the first stage of hydrolysis, each have different focuses and main functions. Bacillus subtilis protease is an basic serine protease with high cleavage activity for peptide bonds following basic amino acid residues such as lysine and arginine, enabling rapid cleavage of the soybean protein backbone. Trypsin specifically recognizes the carboxyl-terminal peptide bonds of lysine and arginine, precisely cleaving these sites. Papain is a cysteine ​​protease with broad-spectrum activity for peptide bonds following hydrophobic amino acid residues such as phenylalanine, leucine, and tryptophan, supplementing the cleavage blind spots of the first two enzymes. The synergistic effect of the three enzymes achieves multi-site, high-efficiency hydrolysis of soybean protein, degrading large protein molecules into medium-molecular-weight peptides. At the same time, due to the different substrate specificities of the three enzymes, the peptide sequence composition and molecular weight distribution of their hydrolysis products exhibit diverse characteristics, creating favorable conditions for the second stage of refined hydrolysis and enrichment of functional peptides. From the perspective of synergistic effects, the deamidation pretreatment of glutamine transaminase increases the charge density and surface hydrophobicity of the protein substrate, enhances the recognition efficiency and binding affinity of the complex protease for the substrate, thereby improving the reaction rate and degree of hydrolysis in the first stage. At the same time, the deamidation treatment reduces the bitter peptide precursor, which works synergistically with the multi-point cleavage strategy of the complex protease to ensure that bitterness is effectively suppressed while achieving deep hydrolysis, ultimately achieving a dual improvement in digestibility and palatability.

[0023] (3) Beneficial technical effects

[0024] 1. Significantly Improved Protein Digestibility and Nutrient Density: This invention utilizes a three-stage targeted enzymatic hydrolysis technology—a pretreatment with glutamine transaminase followed by deamidation, then a first-stage hydrolysis using Bacillus subtilis protease-trypsin-papain, and a second-stage hydrolysis using aminopeptidase-phytase-β-glucosidase—to precisely degrade soy protein isolate into functional peptides with a molecular weight of 200-1500 Da. The peptide mass fraction within this molecular weight range is no less than 70%, significantly higher than the 40-50% level of traditional single-stage or two-stage hydrolysis processes. Simultaneously, free amino nitrogen is increased by no less than 20%, ensuring efficient digestion and absorption of protein by infants and young children. The introduction of phytase degrades phytic acid in soybeans, releasing chelated minerals such as calcium, iron, and zinc, improving mineral bioavailability and achieving a significant increase in nutrient density.

[0025] 2. Effectively suppresses bitterness and improves sensory quality: This invention reduces the content of glutamine residues, the precursor to bitter peptides, at the source through deamidation pretreatment. Combined with three-stage differentiated hydrolysis to precisely regulate peptide sequence composition, it avoids the large-scale generation of bitter peptides with continuous hydrophobic amino acid sequences. At the same time, β-glucosidase hydrolyzes glycosides such as daidzein and other anti-nutritional factors, reducing beany and astringent tastes. This comprehensively improves the palatability of soy-based infant formula powder, allowing the product to maintain good sensory acceptance while undergoing deep hydrolysis. This solves the technical contradiction in traditional hydrolysis processes where deep hydrolysis and bitterness suppression are difficult to achieve simultaneously.

[0026] 3. Achieving high calcium fortification and excellent instant solubility and stability: This invention employs in-situ complexation technology. By introducing carbon dioxide and adding calcium carbonate into the hydrolysate after enzymatic hydrolysis, calcium ions and negatively charged peptide carboxyl groups form stable calcium-peptide chelates in situ in the liquid phase. This avoids the large particle precipitation and clumping problems caused by the subsequent addition of calcium salts. It ensures that the calcium content in the soy milk powder reaches 400-800 mg / 100 g, while the instant solubility time is no more than 30 s and the sedimentation rate is no more than 3% after standing for 30 min at 25℃ and water hardness of 15-200 ppm. This is significantly better than the instant solubility time of 60-90 s and sedimentation rate of 8-12% of the traditional process of adding calcium salts later. The median particle size D50 of the powder is controlled at 50-180 μm and the zeta potential is -10 to -25 mV, ensuring long-term storage stability, and the median particle size drift is less than 15%.

[0027] 4. Ensuring process controllability and batch quality stability: This invention controls the system conductivity to no higher than 2.5 mS / cm by setting a water replenishment or ultrafiltration conductivity regulation step between the two hydrolysis stages. This eliminates the inhibitory effect of salt accumulation on enzyme activity during multi-stage enzymatic hydrolysis, ensuring that each stage of enzymatic reaction proceeds efficiently under optimal ionic strength conditions. This avoids problems such as decreased hydrolysis efficiency, prolonged reaction time, and product quality fluctuations caused by ionic strength accumulation in traditional processes. At the same time, the final heat inactivation step controls the residual enzyme activity to no higher than 5%, preventing the product from continuing to undergo enzymatic reactions during storage. This ensures batch-to-batch quality consistency and long-term product stability, significantly improving the controllability and reproducibility of industrial production.

[0028] 5. Expanding Application Scope and Promoting Technological Progress in the Industry: The soy milk powder prepared by this invention can be used as a core raw material for soy-based infant formula, soy-based follow-up formula, and soy-based toddler formula, etc., accounting for 30-80% of the mass of the formula food. It provides a nutritionally complete, easily digestible and absorbable, and highly sensory-acceptable plant protein substitute for infants with milk protein allergies. At the same time, the three-stage directional enzymatic hydrolysis technology, in-situ complexation technology, and conductivity regulation technology of this invention can be extended to the deep processing of other plant proteins such as pea protein and rice protein, promoting technological progress and product innovation in the plant-based infant nutrition food industry. Attached Figure Description

[0029] Figure 1 This is a molecular weight distribution diagram of Example 1 and Comparative Example 1 of the present invention.

[0030] Figure 2 The image shows the test results (surface, 0 s) of XPS C 1s high-resolution spectrum fitting for Example 1 and Comparative Example 1.

[0031] Figure 3 The results of XPS Ca 2p high-resolution spectra of Examples 1, 2 and Comparative Example 1 (at different sputtering times) are shown.

[0032] Figure 4 The graph shows the test results of FTIR transmission mode and second derivative for Example 1 and Comparative Example 1 (KBr pellet).

[0033] Figure 5 The graph shows the test results of the XPS Ca content-sputtering depth relationship curves for Example 1 and Comparative Example 1. Detailed Implementation

[0034] 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.

[0035] Example 1

[0036] This embodiment provides an enzyme composition for infant soy milk powder and the prepared soy milk powder, comprising, by weight: 5 parts of transglutaminase, 30 parts of Bacillus subtilis protease, 25 parts of trypsin, 15 parts of papain, 12 parts of aminopeptidase, 7 parts of phytase, and 3 parts of β-glucosidase; the aminopeptidase in this embodiment is derived from Aspergillus oryzae.

[0037] In the enzyme composition of this embodiment, the reaction pH of the transglutaminase is 6.4 and the reaction temperature is 40°C; the reaction pH of the Bacillus subtilis protease, trypsin and papain is 7.8 and the reaction temperature is 52°C; the reaction pH of the aminopeptidase, phytase and β-glucosidase is 7.1 and the reaction temperature is 53°C.

[0038] The order of use of the enzyme composition in this embodiment is as follows: first, add the transglutaminase of this embodiment for deamidation pretreatment; then add Bacillus subtilis protease, trypsin, and papain of this embodiment for the first stage of hydrolysis; then add aminopeptidase, phytase, and β-glucosidase of this embodiment for the second stage of hydrolysis. The reaction time for the deamidation pretreatment in this embodiment is 75 min; the reaction time for the first stage of hydrolysis in this embodiment is 75 min; the reaction time for the second stage of hydrolysis in this embodiment is 45 min; the free amino nitrogen after deamidation pretreatment is increased by 25%.

[0039] The specific steps for preparing soy milk powder in this embodiment are as follows:

[0040] S1: Deamide pretreatment

[0041] Take 100 kg of soy protein isolate (protein content ≥90%, analytical grade, commercially available) and add purified water to prepare a 10% (w / w) protein solution. Stir until homogeneous. Adjust the pH to 6.4 with 1 mol / L NaOH solution and heat to 40℃. Add 4500 U / kg of transglutaminase (enzyme activity 200 U / g, food grade, commercially available) per 1000 kg of base protein. React at 40℃ and pH 6.4 for 75 min, stirring every 15 min to maintain homogeneity. After the reaction, raise the temperature to 85℃ and maintain for 5 min to inactivate the enzyme.

[0042] S2: Two-stage directional hydrolysis

[0043] First stage of hydrolysis: The solution obtained in S1 was cooled to 52℃, and the pH was adjusted to 7.8 with 1 mol / L NaOH solution. Based on 1000 kg of base protein, Bacillus subtilis protease (enzyme activity 100,000 U / g, food grade, commercially available), trypsin (enzyme activity 2,500 U / mg, pharmaceutical grade, commercially available), and papain (enzyme activity 600,000 U / g, food grade, commercially available) were added sequentially, with a total enzyme activity of 12,500 U / kg. The reaction was carried out at 52℃ and pH 7.8 for 75 min, with stirring every 20 min. The pH was maintained stable using 1 mol / L NaOH solution during the reaction.

[0044] Second stage hydrolysis: Adjust the pH of the above solution to 7.1 and maintain the temperature at 53℃. Based on 1000 kg of base protein, add aminopeptidase (enzyme activity 50 U / mg, derived from Aspergillus oryzae, food grade, commercially available), phytase (enzyme activity 5000 U / g, food grade, commercially available), and β-glucosidase (enzyme activity 100000 U / g, food grade, commercially available) sequentially, with a total enzyme activity of 6000 U / kg. React at 53℃ and pH 7.1 for 45 min, stirring every 15 min during the reaction.

[0045] S3: Conductivity Regulation

[0046] Between the first and second stages of hydrolysis, the system conductivity was controlled to 2.0 mS / cm (measured with a conductivity meter at 25℃) by adding purified water for dilution.

[0047] S4: In situ complexation

[0048] The pH of the hydrolysate obtained from S2 was adjusted to 5.6, and the temperature was adjusted to 48℃. Food-grade carbon dioxide (purity ≥99.9%, commercially available) was slowly bubbled into the system, while calcium carbonate powder (food-grade, particle size ≤10 μm, commercially available) was added in batches as a calcium source, controlling the molar ratio of peptide carboxyl groups to calcium ions to be 5:1. The mixture was matured at 48℃ and pH 5.6 for 15 min, during which carbon dioxide was continuously bubbled to maintain pH stability. Mechanical stirring (300 rpm) was used during maturation to ensure uniform dispersion of the system.

[0049] S5: Citric Acid Addition

[0050] Add citric acid (food grade, purity ≥99.5%, commercially available) to the complex solution obtained in S4. The molar ratio of citric acid to calcium is 0.2. Maintain the pH value at 5.6 with 1 mol / L NaOH solution. Let it mature at 48℃ for 20 min, stirring (300 rpm) to keep it uniform.

[0051] S6: Terminal heat inactivation and sterilization

[0052] The solution obtained from S5 was heated to 80°C and maintained for 10 min to achieve complete inactivation and sterilization of residual enzyme activity. Continuous stirring (200 rpm) was maintained during the heat treatment to prevent localized overheating.

[0053] S7: Spray drying

[0054] The solution obtained from S6 was fed into a spray drying tower (pressure atomizing nozzle), with the inlet air temperature set at 180℃ and the outlet temperature controlled at 68℃. The atomization pressure was 20 MPa, and the feed rate was 50 kg / h. The spray-dried powder was collected by a cyclone separator to obtain infant soy milk powder. The obtained powder was subjected to a surface instantaneous melting treatment: the powder was placed in a fluidized bed, hot air at 80℃ (relative humidity 15%) was introduced, the treatment time was 2 min, and then it was rapidly cooled to room temperature, bagged, and sealed for storage.

[0055] Features of Example 1

[0056] This embodiment employs a moderately proportioned enzyme composition (the amount of each enzyme is at the middle or lower end of the range of the claims), with mild and moderate reaction conditions (deamidation pH 6.4, temperature 40℃; first-stage hydrolysis pH 7.8, temperature 52℃; second-stage hydrolysis pH 7.1, temperature 53℃), moderate reaction times (deamidation 75 min, first-stage hydrolysis 75 min, second-stage hydrolysis 45 min), and moderate in-situ complexation conditions (pH 5.6, temperature 48℃, peptide carboxyl:Ca molar ratio 5:1, maturation 15 min). The spray drying outlet temperature of 68℃ is within the moderate range. This process features good stability, strong controllability, and high reproducibility, and can produce soy milk powder products with excellent instant solubility, low sedimentation rate, good storage stability, and reasonable peptide distribution. This embodiment is suitable for large-scale production, has high tolerance for process parameter fluctuations, and is suitable as a core protein raw material for infant soy-based formula, follow-up formula, or toddler formula. It is recommended for use in formulations with a soy milk powder content of 50-70%.

[0057] Example 2

[0058] This embodiment provides an enzyme composition for infant soy milk powder and the prepared soy milk powder, comprising, by weight: 2 parts of transglutaminase, 28 parts of Bacillus subtilis protease, 22 parts of trypsin, 12 parts of papain, 11 parts of aminopeptidase, 6 parts of phytase, and 2 parts of β-glucosidase; the aminopeptidase in this embodiment is derived from Aspergillus oryzae.

[0059] In the enzyme composition of this embodiment, the reaction pH of the transglutaminase is 6.2 and the reaction temperature is 38°C; the reaction pH of the Bacillus subtilis protease, trypsin and papain is 7.7 and the reaction temperature is 51°C; the reaction pH of the aminopeptidase, phytase and β-glucosidase is 7.0 and the reaction temperature is 52°C.

[0060] The order of use of the enzyme composition in this embodiment is as follows: first, add the transglutaminase of this embodiment for deamidation pretreatment; then add Bacillus subtilis protease, trypsin, and papain of this embodiment for the first stage of hydrolysis; then add aminopeptidase, phytase, and β-glucosidase of this embodiment for the second stage of hydrolysis. The reaction time for the deamidation pretreatment in this embodiment is 65 min; the reaction time for the first stage of hydrolysis in this embodiment is 85 min; the reaction time for the second stage of hydrolysis in this embodiment is 50 min; the free amino nitrogen after deamidation pretreatment increases by 22%.

[0061] The specific steps for preparing soy milk powder in this embodiment are as follows:

[0062] S1: Deamide pretreatment

[0063] Take 100 kg of soy protein isolate (protein content ≥90%, analytical grade, commercially available) and add purified water to prepare a 10% (w / w) protein solution. Stir until homogeneous. Adjust the pH to 6.2 with 1 mol / L NaOH solution and heat to 38℃. Add 2500 U / kg of transglutaminase (enzyme activity 300 U / g, food grade, commercially available) per 1000 kg of base protein. React at 38℃ and pH 6.2 for 65 min, stirring every 15 min to maintain homogeneity. After the reaction, raise the temperature to 85℃ and maintain for 5 min to inactivate the enzyme.

[0064] S2: Two-stage directional hydrolysis

[0065] First stage of hydrolysis: The solution obtained in S1 was cooled to 51℃, and the pH was adjusted to 7.7 with 1 mol / L NaOH solution. Based on 1000 kg of base protein, Bacillus subtilis protease (enzyme activity 80000 U / g, food grade, commercially available), trypsin (enzyme activity 2500 U / mg, pharmaceutical grade, commercially available), and papain (enzyme activity 600000 U / g, food grade, commercially available) were added sequentially, with a total enzyme activity of 8000 U / kg. The reaction was carried out at 51℃ and pH 7.7 for 85 min, with stirring every 20 min. The pH was maintained stable using 1 mol / L NaOH solution during the reaction.

[0066] Second stage hydrolysis: Adjust the pH of the above solution to 7.0 and maintain the temperature at 52℃. Based on 1000 kg of base protein, add aminopeptidase (enzyme activity 50 U / mg, derived from Aspergillus oryzae, food grade, commercially available), phytase (enzyme activity 5000 U / g, food grade, commercially available), and β-glucosidase (enzyme activity 100000 U / g, food grade, commercially available) sequentially, with a total enzyme activity of 4000 U / kg. React at 52℃ and pH 7.0 for 50 min, stirring every 15 min during the reaction.

[0067] S3: Conductivity Regulation

[0068] Between the first and second stages of hydrolysis, small molecule salt ions were removed by ultrafiltration concentration (molecular weight cutoff 3000 Da), and then purified water was added for dilution to control the conductivity of the system to 1.8 mS / cm (measured by a conductivity meter at 25℃).

[0069] S4: In situ complexation

[0070] The pH of the hydrolysate obtained from S2 was adjusted to 5.5, and the temperature was adjusted to 45℃. Food-grade carbon dioxide (purity ≥99.9%, commercially available) was slowly bubbled into the system, while calcium carbonate powder (food-grade, particle size ≤10 μm, commercially available) was added in batches as a calcium source, controlling the molar ratio of peptide carboxyl groups to calcium ions to be 4:1. The mixture was matured at 45℃ and pH 5.5 for 12 min, during which carbon dioxide was continuously bubbled to maintain pH stability. Mechanical stirring (300 rpm) was used during maturation to ensure uniform dispersion of the system.

[0071] S5: Citric Acid Addition

[0072] Add citric acid (food grade, purity ≥99.5%, commercially available) to the complex solution obtained in S4. The molar ratio of citric acid to calcium is 0.15. Maintain the pH value at 5.5 with 1 mol / L NaOH solution. Let it mature at 45℃ for 15 min, stirring (300 rpm) to keep it uniform.

[0073] S6: Terminal heat inactivation and sterilization

[0074] The solution obtained from S5 was heated to 78°C and maintained for 8 minutes to achieve complete inactivation and sterilization of residual enzyme activity. Continuous stirring (200 rpm) was maintained during the heat treatment to prevent localized overheating.

[0075] S7: Spray drying

[0076] The solution obtained from S6 was fed into a spray drying tower (pressure atomizing nozzle), with the inlet air temperature set at 175℃ and the outlet temperature controlled at 66℃. The atomization pressure was 18 MPa, and the feed rate was 50 kg / h. The spray-dried powder was collected by a cyclone separator to obtain infant soy milk powder. The obtained powder was subjected to a surface instantaneous melting treatment: the powder was placed in a fluidized bed, hot air at 75℃ (relative humidity 20%) was introduced, the treatment time was 1.5 min, and then it was rapidly cooled to room temperature, bagged, and sealed for storage.

[0077] Features of Example 2

[0078] This embodiment uses a relatively low enzyme composition formulation (2 parts of transglutaminase at the lower limit, and the remaining enzymes at medium to low amounts), and relatively mild reaction conditions (deamidation pH 6.2 and temperature 38℃ are relatively low; first-stage hydrolysis pH 7.7 and temperature 51℃ are relatively low; second-stage hydrolysis pH 7.0 is the lower limit, and temperature 52℃). The deamidation time of 65 min is relatively short, while the first-stage hydrolysis time of 85 min is relatively long to compensate for the low enzyme dosage. The second-stage hydrolysis time of 50 min is relatively long. The in-situ complexation conditions are relatively mild (pH 5.5, temperature 45℃, peptide carboxyl:Ca molar ratio 4:1, maturation 12 min), and the spray drying outlet temperature of 66℃ is relatively low. This process, by extending the hydrolysis time and optimizing the complexation conditions, can achieve good product quality while reducing enzyme dosage and energy consumption, making it suitable for applications with strict cost control. This embodiment is applicable to infant soy-based formula products with moderate requirements for instant solubility and a greater emphasis on cost-effectiveness. It is recommended to use it in formulas with a soy milk powder content of 40-60%.

[0079] Example 3

[0080] This embodiment provides an enzyme composition for infant soy milk powder and the soy milk powder prepared therefrom, comprising, by weight: 8 parts of transglutaminase, 33 parts of Bacillus subtilis protease, 28 parts of trypsin, 18 parts of papain, 14 parts of aminopeptidase, 9 parts of phytase, and 4.5 parts of β-glucosidase; the aminopeptidase in this embodiment is derived from Aspergillus oryzae.

[0081] In the enzyme composition of this embodiment, the reaction pH of the transglutaminase is 6.7 and the reaction temperature is 44°C; the reaction pH of the Bacillus subtilis protease, trypsin and papain is 7.9 and the reaction temperature is 54°C; the reaction pH of the aminopeptidase, phytase and β-glucosidase is 7.2 and the reaction temperature is 55°C.

[0082] The order of use of the enzyme composition in this embodiment is as follows: first, add the transglutaminase of this embodiment for deamidation pretreatment; then add Bacillus subtilis protease, trypsin, and papain of this embodiment for the first stage of hydrolysis; then add aminopeptidase, phytase, and β-glucosidase of this embodiment for the second stage of hydrolysis. The reaction time for the deamidation pretreatment in this embodiment is 85 min; the reaction time for the first stage of hydrolysis in this embodiment is 65 min; the reaction time for the second stage of hydrolysis in this embodiment is 35 min; the free amino nitrogen after deamidation pretreatment is increased by 28%.

[0083] The specific steps for preparing soy milk powder in this embodiment are as follows:

[0084] S1: Deamide pretreatment

[0085] Take 100 kg of soy protein isolate (protein content ≥90%, analytical grade, commercially available) and add purified water to prepare a 10% (w / w) protein solution. Stir until homogeneous. Adjust the pH to 6.7 with 1 mol / L NaOH solution and heat to 44℃. Add 7500 U / kg of transglutaminase (enzyme activity 400 U / g, food grade, commercially available) per 1000 kg of base protein. React at 44℃ and pH 6.7 for 85 min, stirring every 15 min to maintain homogeneity. After the reaction, raise the temperature to 85℃ and maintain for 5 min to inactivate the enzyme.

[0086] S2: Two-stage directional hydrolysis

[0087] First stage of hydrolysis: The solution obtained in S1 was cooled to 54℃, and the pH was adjusted to 7.9 with 1 mol / L NaOH solution. Based on 1000 kg of base protein, Bacillus subtilis protease (enzyme activity 150,000 U / g, food grade, commercially available), trypsin (enzyme activity 2,500 U / mg, pharmaceutical grade, commercially available), and papain (enzyme activity 600,000 U / g, food grade, commercially available) were added sequentially, with a total enzyme activity of 18,000 U / kg. The reaction was carried out at 54℃ and pH 7.9 for 65 min, with stirring every 20 min. The pH was maintained stable using 1 mol / L NaOH solution during the reaction.

[0088] Second stage hydrolysis: Adjust the pH of the above solution to 7.2 and maintain the temperature at 55℃. Based on 1000 kg of base protein, add aminopeptidase (enzyme activity 50 U / mg, derived from Aspergillus oryzae, food grade, commercially available), phytase (enzyme activity 5000 U / g, food grade, commercially available), and β-glucosidase (enzyme activity 100000 U / g, food grade, commercially available) sequentially, with a total enzyme activity of 9000 U / kg. React at 55℃ and pH 7.2 for 35 min, stirring every 15 min during the reaction.

[0089] S3: Conductivity Regulation

[0090] Between the first and second stages of hydrolysis, the system conductivity was controlled to 2.3 mS / cm (measured with a conductivity meter at 25℃) by adding purified water for dilution.

[0091] S4: In situ complexation

[0092] The pH of the hydrolysate obtained from S2 was adjusted to 5.7, and the temperature was adjusted to 52℃. Food-grade carbon dioxide (purity ≥99.9%, commercially available) was slowly bubbled into the system, while calcium carbonate powder (food-grade, particle size ≤10 μm, commercially available) was added in batches as a calcium source, controlling the molar ratio of peptide carboxyl groups to calcium ions to be 7:1. The mixture was matured at 52℃ and pH 5.7 for 18 min, with continuous bubbling of carbon dioxide to maintain pH stability. Mechanical stirring (300 rpm) was used during maturation to ensure uniform dispersion of the system.

[0093] S5: Citric Acid Addition

[0094] Add citric acid (food grade, purity ≥99.5%, commercially available) to the complex solution obtained in S4. The molar ratio of citric acid to calcium is 0.25. Maintain the pH value at 5.7 with 1 mol / L NaOH solution. Let it mature at 52℃ for 25 min, stirring (300 rpm) to keep it uniform.

[0095] S6: Terminal heat inactivation and sterilization

[0096] The solution obtained from S5 was heated to 83°C and maintained for 12 min to achieve complete inactivation and sterilization of residual enzyme activity. Continuous stirring (200 rpm) was maintained during the heat treatment to prevent localized overheating.

[0097] S7: Spray drying

[0098] The solution obtained from S6 was fed into a spray drying tower (pressure atomizing nozzle), with the inlet air temperature set at 185℃ and the outlet temperature controlled at 70℃. The atomization pressure was 23 MPa, and the feed rate was 50 kg / h. The spray-dried powder was collected by a cyclone separator to obtain infant soy milk powder. The obtained powder was subjected to a surface instantaneous melting treatment: the powder was placed in a fluidized bed, hot air at 85℃ (relative humidity 10%) was introduced, the treatment time was 2.5 min, and then it was rapidly cooled to room temperature, bagged, and sealed for storage.

[0099] Features of Example 3

[0100] This embodiment employs a relatively high enzyme composition formulation (8 parts of transglutaminase at the upper limit, 33 parts of Bacillus subtilis protease and 14 parts of aminopeptidase close to the upper limit, and 4.5 parts of β-glucosidase close to the upper limit), with intensified reaction conditions (deamidation pH 6.7 and temperature 44℃ are relatively high; first-stage hydrolysis pH 7.9 and temperature 54℃ are relatively high; second-stage hydrolysis pH 7.2 and temperature 55℃ are at the upper limit), a relatively long deamidation time of 85 min, while the first and second-stage hydrolysis times are moderate or relatively short to avoid over-hydrolysis, and in-situ complexation conditions are relatively intensified (pH 5.7, temperature 52℃ are relatively high, peptide carboxyl:Ca molar ratio 7:1 is relatively high, and maturation time 18 min is relatively long), with the spray drying outlet temperature of 70℃ at the upper limit. This process, through high enzyme content and intensified conditions, achieves rapid and efficient deamidation and hydrolysis, yielding a high-quality soy milk powder product with a large increase in free amino nitrogen, narrow peptide molecular weight distribution, excellent solubility, and extremely low sedimentation rate. This embodiment is applicable to high-end infant soy-based formula powder products that require high quality, excellent solubility and low sedimentation rate, and is recommended for use in formulas with a soy milk powder content of 60-80%.

[0101] Example 4

[0102] This embodiment provides an enzyme composition for infant soy milk powder and the prepared soy milk powder, comprising, by weight: 3 parts of transglutaminase, 25 parts of Bacillus subtilis protease, 20 parts of trypsin, 10 parts of papain, 10 parts of aminopeptidase, 5 parts of phytase, and 1 part of β-glucosidase; the aminopeptidase in this embodiment is derived from Aspergillus oryzae.

[0103] In the enzyme composition of this embodiment, the reaction pH of the transglutaminase is 6.0 and the reaction temperature is 35°C; the reaction pH of the Bacillus subtilis protease, trypsin and papain is 7.6 and the reaction temperature is 50°C; the reaction pH of the aminopeptidase, phytase and β-glucosidase is 7.05 and the reaction temperature is 52.5°C.

[0104] The order of use of the enzyme composition in this embodiment is as follows: first, add the transglutaminase of this embodiment for deamidation pretreatment; then add Bacillus subtilis protease, trypsin, and papain of this embodiment for the first stage of hydrolysis; then add aminopeptidase, phytase, and β-glucosidase of this embodiment for the second stage of hydrolysis. The reaction time for the deamidation pretreatment in this embodiment is 60 min; the reaction time for the first stage of hydrolysis in this embodiment is 90 min; the reaction time for the second stage of hydrolysis in this embodiment is 60 min; the free amino nitrogen after deamidation pretreatment is increased by 20%.

[0105] The specific steps for preparing soy milk powder in this embodiment are as follows:

[0106] S1: Deamide pretreatment

[0107] Take 100 kg of soy protein isolate (protein content ≥90%, analytical grade, commercially available) and add purified water to prepare a 10% (w / w) protein solution. Stir until homogeneous. Adjust the pH to 6.0 with 1 mol / L NaOH solution and heat to 35℃. Add 1500 U / kg of transglutaminase (enzyme activity 100 U / g, food grade, commercially available) per 1000 kg of base protein. React at 35℃ and pH 6.0 for 60 min, stirring every 15 min to maintain homogeneity. After the reaction, raise the temperature to 85℃ and maintain for 5 min to inactivate the enzyme.

[0108] S2: Two-stage directional hydrolysis

[0109] First stage of hydrolysis: The solution obtained in S1 was cooled to 50℃, and the pH was adjusted to 7.6 with 1 mol / L NaOH solution. Based on 1000 kg of base protein, Bacillus subtilis protease (enzyme activity 50000 U / g, food grade, commercially available), trypsin (enzyme activity 2500 U / mg, pharmaceutical grade, commercially available), and papain (enzyme activity 600000 U / g, food grade, commercially available) were added sequentially, with a total enzyme activity of 5000 U / kg. The reaction was carried out at 50℃ and pH 7.6 for 90 min, with stirring every 20 min. The pH was maintained stable using 1 mol / L NaOH solution during the reaction.

[0110] Second stage hydrolysis: Adjust the pH of the above solution to 7.05 and maintain the temperature at 52.5℃. Based on 1000 kg of base protein, add aminopeptidase (enzyme activity 50 U / mg, derived from Aspergillus oryzae, food grade, commercially available), phytase (enzyme activity 5000 U / g, food grade, commercially available), and β-glucosidase (enzyme activity 100000 U / g, food grade, commercially available) sequentially, with a total enzyme activity of 2000 U / kg. React at 52.5℃ and pH 7.05 for 60 min, stirring every 15 min during the reaction.

[0111] S3: Conductivity Regulation

[0112] Between the first and second stages of hydrolysis, small molecule salt ions were removed by ultrafiltration concentration (molecular weight cutoff 3000 Da), and then purified water was added to dilute the system, controlling the conductivity of the system to 1.5 mS / cm (measured by a conductivity meter at 25℃).

[0113] S4: In situ complexation

[0114] The pH of the hydrolysate obtained from S2 was adjusted to 5.4, and the temperature was adjusted to 40℃. Food-grade carbon dioxide (purity ≥99.9%, commercially available) was slowly bubbled into the system, while calcium carbonate powder (food-grade, particle size ≤10 μm, commercially available) was added in batches as a calcium source, controlling the molar ratio of peptide carboxyl groups to calcium ions to be 3:1. The mixture was matured at 40℃ and pH 5.4 for 10 min, during which carbon dioxide was continuously bubbled to maintain pH stability. Mechanical stirring (300 rpm) was used during maturation to ensure uniform dispersion of the system.

[0115] S5: Citric Acid Addition

[0116] Add citric acid (food grade, purity ≥99.5%, commercially available) to the complex solution obtained in S4. The molar ratio of citric acid to calcium is 0.1. Maintain the pH value at 5.4 with 1 mol / L NaOH solution. Let it mature at 40℃ for 10 min, stirring (300 rpm) to keep it uniform.

[0117] S6: Terminal heat inactivation and sterilization

[0118] The solution obtained from S5 was heated to 75°C and maintained for 15 min to achieve complete inactivation and sterilization of residual enzyme activity. Continuous stirring (200 rpm) was maintained during the heat treatment to prevent localized overheating.

[0119] S7: Spray drying

[0120] The solution obtained from S6 was fed into a spray drying tower (pressure atomizing nozzle), with the inlet air temperature set at 170℃ and the outlet temperature controlled at 65℃. The atomization pressure was 16 MPa, and the feed rate was 50 kg / h. The spray-dried powder was collected by a cyclone separator to obtain infant soy milk powder. The obtained powder was subjected to a surface instantaneous melting treatment: the powder was placed in a fluidized bed, hot air at 70℃ (relative humidity 25%) was introduced, the treatment time was 1 min, and it was rapidly cooled to room temperature, then bagged and sealed for storage.

[0121] Features of Example 4

[0122] This embodiment employs a formulation and process with multiple boundary verification strategies: multiple components in the enzyme composition formulation are set to their lower limits (3 parts glutamine transaminase, 25 parts Bacillus subtilis protease, 20 parts trypsin, 10 parts papain, 10 parts aminopeptidase, 5 parts phytase, and 1 part β-glucosidase); multiple reaction conditions are set to their boundary values ​​(deamidation pH 6.0, temperature 35℃; first-stage hydrolysis pH 7.6, temperature 50℃; deamidation time 60 min, first-stage hydrolysis time 90 min, and second-stage hydrolysis time 60 min); multiple in-situ complexation conditions are set to their boundary values ​​(pH 5.4, temperature 40℃, peptide carboxyl:Ca molar ratio 3:1, ripening for 10 min, citric acid:Ca molar ratio 0.1, citric acid ripening for 10 min); thermal inactivation conditions are set to their boundary values ​​(temperature 75℃, time 15 min); and the spray drying outlet temperature is 65℃. This process, by verifying multiple boundary values ​​within the scope of the claims, demonstrates that the technical solution remains feasible even under extreme conditions such as minimum enzyme dosage, mild reaction conditions, and low-energy complexation drying. It can yield soy milk powder products that meet quality standards, fully supporting the boundary feasibility of the claims. This embodiment is applicable to scenarios with extremely high cost control requirements, process tolerance assessment, and boundary condition verification, providing crucial data support for the boundary rationality of the claims.

[0123] Comparative Example 1

[0124] It is basically the same as Example 1, except that the amount of transglutaminase is 1 part, while the amounts of other components and preparation conditions remain unchanged.

[0125] Comparative Example 2

[0126] The preparation method is basically the same as in Example 1, except that the amount of transglutaminase used is 10 parts, while the amounts of other components and preparation conditions remain unchanged.

[0127] Comparative Example 3

[0128] The preparation method is basically the same as in Example 1, except that the amount of Bacillus subtilis protease used is 22 parts, while the amounts of other components and preparation conditions remain unchanged.

[0129] Comparative Example 4

[0130] It is basically the same as Example 1, except that the amount of β-glucosidase is 6 parts, while the amount of other components and preparation conditions remain unchanged.

[0131] Comparative Example 5

[0132] The process is basically the same as in Example 1, except that the reaction temperature for the deamidation pretreatment is 32°C, while other conditions remain unchanged.

[0133] Comparative Example 6

[0134] The process is basically the same as in Example 1, except that the reaction temperature for the deamidation pretreatment is 48°C, while other conditions remain unchanged.

[0135] Comparative Example 7

[0136] It is basically the same as Example 1, except that the pH value of the first stage of hydrolysis is 7.4, while other conditions remain unchanged.

[0137] Comparative Example 8

[0138] It is basically the same as Example 1, except that the reaction time of the first stage of hydrolysis is 55 min, and other conditions remain unchanged.

[0139] Comparative Example 9

[0140] The method is basically the same as in Example 1, except that the conductivity regulation step (S3) is omitted and the in-situ complexation is directly entered. The conductivity of the system is 3.8 mS / cm, and other conditions remain unchanged.

[0141] Comparative Example 10

[0142] It is basically the same as Example 1, except that the pH value of the in-situ complexation is 5.2, and other conditions remain unchanged.

[0143] Comparative Example 11

[0144] The procedure is basically the same as in Example 1, except that the molar ratio of the in-situ complexed peptide carboxyl group to calcium ion is 2:1, while other conditions remain unchanged.

[0145] Comparative Example 12

[0146] The procedure is basically the same as in Example 1, except that the citric acid addition step (S5) is omitted and thermal deactivation is performed directly, while other conditions remain unchanged.

[0147] Comparative Example 13

[0148] It is basically the same as Example 1, except that the spray drying outlet temperature is 72°C, while other conditions remain unchanged.

[0149] Performance testing:

[0150] Experiment 1: Determination of peptide molecular weight distribution

[0151] Test subject: Reconstituted soy milk powder solutions prepared in the examples and comparative examples

[0152] Test objective: To evaluate the effect of enzymatic hydrolysis process on peptide molecular weight distribution and verify that the proportion of peptides in the 200-1500 Da range is ≥70% (claim 5).

[0153] Test principle: High-performance gel permeation chromatography (GPC) separates peptides based on molecular size, and quantifies them using a UV detector (220 nm). A molecular weight-retention time calibration curve is established using a mixture of standard peptides.

[0154] Experimental methods:

[0155] Accurately weigh 1.0 g of soy milk powder sample into a 50 mL volumetric flask, dissolve and dilute to volume with purified water, and filter through a 0.45 μm filter membrane. HPLC conditions: TSKgel G2000SWXL column (300 mm × 7.8 mm, 5 μm), mobile phase: 0.1 mol / L phosphate buffer (pH 7.0) + 0.3 mol / L NaCl, flow rate: 0.5 mL / min, column temperature: 30℃, injection volume: 20 μL, detection wavelength: 220 nm. Calibration curves were established using cytochrome C (12384 Da), bacitracin (1423 Da), glycine-glycine-tyrosine-arginine (GGYR, 451 Da), and glycine-glycine-glycine (GGG, 189 Da).

[0156] Standard basis: Refer to GB 5009.5 or similar methods for the determination of molecular weight distribution of protein hydrolysates in food.

[0157] Key parameters: column temperature 30℃, flow rate 0.5 mL / min, detection wavelength 220 nm

[0158] Data processing: Integrate the peak areas for each molecular weight range, and calculate the mass percentage of the total peak area in the 200-1500 Da range.

[0159] Experiment 2: Determination of Dissolution Time and Sedimentation Rate

[0160] Test subjects: Soy milk powder prepared in the examples and comparative examples

[0161] Test objective: To evaluate the dissolving performance and verify that the instant dissolving time is ≤30 s and the sedimentation rate after standing for 30 min is ≤3%.

[0162] Test principle: Simulate actual reconstitution conditions, observe powder dissolution time and particle sedimentation behavior, and quantify reconstitution quality.

[0163] Experimental methods:

[0164] (1) Instant dissolution time: Take 12.5 g of soy milk powder into a 250 mL beaker, add 100 mL of purified water preheated to 25℃ (adjust the water hardness to 100 ppm, add CaCl2 and MgSO4), stir with a glass rod at a constant speed (60 times / min), and record the time (s) when the powder is completely dissolved and there are no visible particles.

[0165] (2) Sedimentation rate: Transfer the above-mentioned reconstituted solution to a 100 mL graduated cylinder, let it stand for 30 min, and measure the volume of the bottom sediment layer V1 (mL) and the total volume V2 (mL). Sedimentation rate = (V1 / V2) × 100%

[0166] Standard basis: Refer to GB / T 18738 Determination method for instant soy powder or similar instant food.

[0167] Key parameters: water temperature 25±1℃, water hardness 100±10 ppm, stirring speed 60 times / min, settling time 30min

[0168] Data processing: Each sample was measured three times, and the mean ± standard deviation was taken.

[0169] Experiment 3: Calcium content determination

[0170] Test subjects: Soy milk powder prepared in the examples and comparative examples

[0171] Test objective: To verify the calcium fortification effect of the in-situ complexation process and evaluate the calcium content (400-800 mg / 100 g) (further limited).

[0172] Test principle: Atomic absorption spectrometry (AAS) quantifies calcium by measuring the absorbance at a characteristic wavelength (422.7 nm).

[0173] Experimental methods:

[0174] Accurately weigh 2.0 g of soy milk powder sample into a crucible, ashing at 550℃ for 4 h until completely carbonized. After cooling, add 10 mL of 6 mol / L hydrochloric acid to dissolve the ash, transfer to a 50 mL volumetric flask and dilute to volume. Measure using a flame atomic absorption spectrometer (FAAS): air-acetylene flame, wavelength 422.7 nm, slit 0.5 nm, lamp current 5 mA. Establish calibration curves using calcium standard solutions (0, 2, 5, 10, 20 mg / L).

[0175] Standard basis: GB 5009.92 Determination of calcium in food

[0176] Key parameters: ashing temperature 550℃, measurement wavelength 422.7 nm, flame type air-acetylene.

[0177] Data processing: Calcium content (mg / 100 g) = (C×V×D) / (m×1000)×100, where C is the concentration to be measured (mg / L), V is the final volume (mL), D is the dilution factor, and m is the sample mass (g).

[0178] Experiment 4: Measurement of zeta potential

[0179] Test subject: Reconstituted soy milk powder solutions prepared in the examples and comparative examples

[0180] Test objective: To evaluate the surface charge and dispersion stability of particles, and to verify the zeta potential from -10 to -25 mV.

[0181] Test principle: Dynamic light scattering technique is used to determine the electrophoretic mobility of particles in an electric field, and the zeta potential is calculated using the Smoluchowski equation.

[0182] Experimental methods:

[0183] Accurately weigh 0.1 g of soy milk powder into a 50 mL beaker, add 10 mL of purified water, and magnetically stir for 5 min to ensure thorough dispersion. Transfer 1 mL of the dispersion to a Malvern Zetasizer Nano ZS folded capillary electrophoresis tank, equilibrate at 25℃ for 3 min, and then measure. Each sample was automatically measured 3 times, with 11 sub-runs per measurement, and the average value was taken.

[0184] Standard basis: Refer to ISO 13099-2 Determination of zeta potential in colloidal systems or similar methods.

[0185] Key parameters: Measurement temperature 25℃, dispersion medium pH 6.5-7.0, sample concentration 1% (w / v)

[0186] Data processing: Automatically output the mean ± standard deviation of the ζ potential (mV) to evaluate dispersion stability (|ζ|>15 mV is considered good).

[0187] Experiment 5: Storage Stability Test

[0188] Test subjects: Soy milk powder prepared in the examples and comparative examples

[0189] Test objective: To evaluate the physical stability of powder under accelerated storage conditions and verify that particle size drift is <15% (claim 5).

[0190] Test principle: After storage at 40℃ / 75% RH for 4-8 weeks, the change in the median particle size D50 of the powder is monitored to reflect the degree of particle agglomeration.

[0191] Experimental methods:

[0192] Soy milk powder samples were dispensed into sealed bags (50 g / bag) and placed in a constant temperature and humidity chamber (40±2℃, relative humidity 75±5%). Samples were taken at 0, 2, 4, 6, and 8 weeks, and the median particle size (D50) was determined using a laser particle size analyzer (Malvern Mastersizer 3000). Anhydrous ethanol was used as the dispersion medium, and the mixture was ultrasonically dispersed for 30 s with a light-blocking rate of 10-15%. The average value of three measurements was taken.

[0193] Standard basis: Refer to GB / T 21924 Determination of Particle Size Distribution of Powder or similar methods

[0194] Key parameters: storage temperature 40±2℃, relative humidity 75±5%, measurement wavelength red light (633 nm) + blue light (470 nm)

[0195] Data processing: Particle size drift rate = (D508 weeks - D500 weeks) / D500 weeks × 100%, used to evaluate storage stability (drift <15% is acceptable).

[0196] Experiment 6: Sensory Evaluation

[0197] Test subjects: Soy milk powder preparation solutions prepared in the examples and comparative examples

[0198] Test objective: To evaluate the sensory quality of the product and verify the effect of enzymatic hydrolysis on improving palatability.

[0199] Testing principle: Trained sensory evaluators conduct a comprehensive evaluation based on color, odor, taste, and texture according to scoring criteria.

[0200] Experimental methods:

[0201] The sample solution was prepared by mixing 12.5 g powder with 100 mL of water (40℃) and cooling to room temperature. Blind evaluation was conducted by 10 trained sensory evaluators (compliant with GB / T 16291.1). Evaluation indicators and scores (total 100 points): color uniformity (20 points), beany intensity (25 points, lower is better), bitterness intensity (25 points, lower is better), texture smoothness (20 points), and overall acceptability (10 points). A 9-point preference scale (1 = extremely disliked, 9 = extremely liked) was used to assist in the scoring.

[0202] Standard basis: GB / T 16291.1 General Rules for Sensory Analysis

[0203] Key parameters: mixing temperature 40℃, sample temperature 25℃, evaluation ambient temperature 20-25℃, white light illumination.

[0204] Data processing: Calculate the average score ± standard deviation of each indicator. A total score ≥80 is considered excellent, and <70 is considered unqualified.

[0205] Figure 1 This study visually demonstrates the decisive impact of deamidation pretreatment on peptide molecular weight distribution: In Example 1, after treatment with glutamine transaminase, GPC chromatography showed concentrated and symmetrical Gaussian peaks in the 200-1500 Da range, with a cumulative mass fraction of 75.2% in this range. The sharp peak shape indicates a narrow and controllable molecular weight distribution. In contrast, in Comparative Example 1, the omission of the deamidation step resulted in insufficient activation of glutamine residues in soybean protein, leading to reduced recognition efficiency of subsequent alkaline and neutral proteases at their cleavage sites. This resulted in a significant increase in the residual high molecular weight peptides >1500 Da, which was manifested in the GPC spectrum as a significantly increased tailing peak area in the early retention time (corresponding to the high molecular weight region), and a decrease in the target region of 200-1500 Da. The proportion of free amino acid content was only 58.5%, which was 116.7 percentage points lower than that of Example 1. Meanwhile, Comparative Example 1 also showed abnormal peak clusters in the ultra-low molecular weight region of <200 Da, indicating that poor enzymatic selectivity led to the over-hydrolysis of some peptides into free amino acids. This bimodal distribution characteristic of "more at both ends and less in the middle" is in stark contrast to the single-peak concentrated distribution of Example 1. This fully demonstrates that the deamidation pretreatment creates a precise enzymatic microenvironment for subsequent two-stage targeted hydrolysis by changing the side chain charge distribution of the substrate and optimizing the conformational flexibility of the protein, ultimately achieving efficient enrichment of the target peptide. This result is corroborated by the free amino content determination and sensory evaluation data, which together support the technical necessity of deamidation pretreatment in the three-stage precise enzymatic hydrolysis process.

[0206] Figure 4 The graph shows the test results (KBr pellet) of FTIR transmission mode and second derivative for Example 1 and Comparative Example 1. Figure 4 As can be seen, in Example 1 at 1730 cm -1 The intensity of the C=O stretching vibration peak at this location increases and the half-width at half-maximum narrows, from 1240 to 1260 cm⁻¹. -1 With 1,080–1,150 cm -1 C–O–C / PO2 interval - After resolution by the second derivative, the relevant peaks exhibit clearer shoulder peaks and a higher peak intensity ratio, while the 1600–1650 cm⁻¹ peaks show a higher intensity ratio. -1 The separation of amide I / conjugated C=C components increases and is accompanied by 3200–3600 cm⁻¹. -1 The narrowing of the hydrogen-bonded association bands and the overall redshift indicate a more ordered coordination and cross-linking environment after the introduction of the target functional group, with a decrease in free hydroxyl groups and an increase in the proportion of coordinated carbonyl groups. This infrared evidence is consistent with... Figure 2 The increase in the proportion of the C=O / C–O component in the test results (surface, 0 s) of XPSC 1s high-resolution spectrum fitting of Example 1 and Comparative Example 1 is consistent with the trend. Figure 3The XPS Ca 2p high-resolution spectra of Examples 1, 2, and Comparative Example 1 (at different sputtering times) show the enhancement of Ca–O coordination components and... Figure 5 The test results of the XPS Ca content-sputtering depth relationship curves of Example 1 and Comparative Example 1 mutually corroborate the establishment of the Ca content gradient from the surface to the near-surface layer, comprehensively proving that the scheme is reasonable and feasible in terms of chemical configuration regulation and interface Ca coordination construction.

[0207] The performance of the examples and comparative examples is summarized in Table 1. The performance differences between the examples and comparative examples stem from the systematic impact of deviations in process parameters on peptide structure distribution, complexation efficiency, and particle stability. Specifically, insufficient transglutaminase or incomplete three-stage enzymatic hydrolysis led to a decrease in the proportion of 200-1500 Da peptides to 58.5-68.5%, lower than the 70.2-78.8% in the examples. Consequently, the residual large-molecule peptides resulted in a stronger beany odor and decreased solubility. Excessively high conductivity, deviating complexation pH from the optimal value, or an excessively low molar ratio caused the peptide-Ca ratio to decrease. 2+ Insufficient complexation led to a decrease in calcium content to 480-630 mg / 100g and a reduction in the absolute value of particle zeta potential to -9.5 to -16.5 mV. Insufficient electrostatic repulsion caused agglomeration and an increase in sedimentation rate to 3.2-8.5%. Omitting citric acid stabilizer resulted in severe particle agglomeration during storage due to the lack of steric protection, with particle size drift increasing dramatically to 35.8% and sedimentation rate as high as 8.5%, while also deteriorating reconstitution properties. Excessive hydrolysis or improper enzymatic hydrolysis conditions produced bitter peptides, and pH deviation from the isoelectric point further weakened surface charge stability. Overall, this resulted in a decrease in the total sensory score of the comparative example to 55-78 points, significantly lower than the 82-92 points of the example. Although the excessive spray drying temperature in Comparative Example 13 had a relatively small impact on other properties, the increased energy consumption weakened the process economy. Overall, this reflects the key role of precise control of process parameters in optimizing peptide distribution, improving complexation efficiency, enhancing particle stability, and improving sensory quality.

[0208] Table 1. Comparison of performance data between the examples and comparative examples

[0209] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. 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. An enzyme composition specifically for infant soy milk powder, characterized in that, The product comprises, by weight, 2-8 parts of transglutaminase, 25-35 parts of Bacillus subtilis protease, 20-30 parts of trypsin, 10-20 parts of papain, 10-15 parts of aminopeptidase, 5-10 parts of phytase, and 1-5 parts of β-glucosidase; wherein the aminopeptidase is derived from Aspergillus oryzae.

2. The enzyme composition according to claim 1, characterized in that, The reaction of the transglutaminase is at a pH of 6.0-6.8 and a reaction temperature of 35-45℃; the reaction of the Bacillus subtilis protease, trypsin and papain is at a pH of 7.6-8.0 and a reaction temperature of 50-55℃; the reaction of the aminopeptidase, phytase and β-glucosidase is at a pH of 7.0-7.2 and a reaction temperature of 52-55℃.

3. The enzyme composition according to claim 1, characterized in that, The order of use of the enzyme composition is as follows: first, add the transglutaminase for deamidation pretreatment; then add the Bacillus subtilis protease, trypsin, and papain for the first stage of hydrolysis; and then add the aminopeptidase, phytase, and β-glucosidase for the second stage of hydrolysis. The reaction time for the deamidation pretreatment is 60-90 min; the reaction time for the first stage of hydrolysis is 60-90 min; and the reaction time for the second stage of hydrolysis is 30-60 min. The free amino nitrogen after deamidation pretreatment is increased by no less than 20%.

4. A soy milk powder, characterized in that, The soy milk powder is prepared by enzymatic hydrolysis using soy protein isolate as raw material, and by in-situ complexation and spray drying using the enzyme composition for infant soy milk powder as described in any one of claims 1-3; the median particle size D50 of the soy milk powder is 50-180 μm, and the water activity is not higher than 0.30; when prepared by reconstitution at a temperature of 25℃ and a water hardness of 15-200 ppm, the instant dissolution time is not higher than 30 s, and the sedimentation rate after standing for 30 min is not higher than 3%.

5. The soy milk powder according to claim 4, characterized in that, The zeta potential is -10 to -25 mV. When stored at 40℃ and 75% relative humidity for 4-8 weeks, the median particle diameter drift is less than 15%, and the mass fraction of peptides with molecular weight of 200-1500 Da is not less than 70%.

6. The method for preparing soy milk powder according to claim 4 or 5, characterized in that, Includes the following steps: S1: Made from soy protein isolate, deamidated using transglutaminase; S2: Two-stage targeted hydrolysis is carried out using an enzyme composition specifically for infant soy milk powder. In the first stage, Bacillus subtilis protease, trypsin and papain are added. In the second stage, aminopeptidase, phytase and β-glucosidase are added. S3: Between the two sections of S2, the conductivity of the system is controlled to be no higher than 2.5 mS / cm by water replenishment or ultrafiltration; S4: Introduce carbon dioxide to perform in-situ complexation using calcium carbonate as a calcium source; S5: Add citric acid; S6: Set the terminal heat inactivation and sterilization steps; S7: Spray drying is used.

7. The method for preparing soy milk powder according to claim 6, characterized in that, The reaction conditions for transglutaminase in S1 are: pH 6.0-6.8, temperature 35-45℃, time 60-90 min, and dosage 1000-8000 U / kg base protein; the reaction conditions for the first stage in S2 are: pH 7.6-8.0, temperature 50-55℃, time 60-90 min, and total enzyme activity dosage 5000-20000 U / kg base protein; the reaction conditions for the second stage are: pH 7.0-7.2, temperature 52-55℃, time 30-60 min, and total enzyme activity dosage 2000-10000 U / kg base protein.

8. The method for preparing soy milk powder according to claim 6 or 7, characterized in that, The reaction conditions in S4 are: pH 5.4-5.8, temperature 40-55℃, molar ratio of peptide carboxyl groups to calcium ions 3-8:1, and ripening time 10-20 min; in S5, the molar ratio of citric acid to calcium is 0.1-0.3, the pH is maintained at 5.4-5.8, and the ripening time is 10-30 min; in S6, the temperature is heated to 75-85℃ and maintained for 5-15 min; in S7, the outlet temperature is 65-70℃, and instantaneous melting treatment of the powder surface is performed.

9. The application of the enzyme composition according to claim 1 in the preparation of soy milk powder, wherein the preparation process includes deamidation pretreatment, two-stage directional hydrolysis, in-situ complexation and spray drying steps; the median particle size D50 of the soy milk powder is 50-180 μm, the water activity is not higher than 0.30, and when prepared at a temperature of 25℃ and a water hardness of 15-200 ppm, the instant dissolution time is not higher than 30 s, and the sedimentation rate after standing for 30 min is not higher than 3%.

10. The application of the soy milk powder according to claim 4 in the preparation of instant nutritional food, wherein the nutritional food includes soy-based infant formula powder, soy-based follow-up formula powder, or soy-based toddler formula powder; the mass percentage of the soy milk powder in the instant nutritional food is 30-80%.

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