Enzymatic modification of foreign protein and application of foreign protein in baked food

Abstract

The invention relates to the technical field of protein processing, and discloses enzymolysis modification of foreign protein and application of the foreign protein in baked foods.The method comprises the steps that before an enzymolysis reaction starts, the foreign protein, proteolytic enzyme, reducing sugar in a specific proportion and food-grade non-reducing polysaccharide are mixed in advance; according to the preparation method, a reducing sugar is added, so that new amino groups generated by cutting off peptide bonds through enzymolysis are covalently anchored by the reducing sugar at the moment of generation, and meanwhile, the chemical anchoring rate is always higher than the physical aggregation rate through physical retardation formed by diffusion of polysaccharide to hydrophobic peptide fragments, and the traditional process route of first degradation and then repair is avoided; the degradation process of the protein is converted into an integrated process which is synchronously carried out with functional modification of the peptide fragment, so that the problems of aggregation and bitter taste of the product are avoided from a molecular source, and the functional protein hydrolysate which can be directly applied to the field of baked foods is obtained.

Description

Technical Field

[0001] This invention relates to the enzymatic modification of exogenous proteins and their application in baked goods, belonging to the field of protein processing technology. Background Technology

[0002] Currently, in the functional modification of proteins, proteases are commonly used to hydrolyze proteins, degrading them into smaller peptides or oligopeptides to improve their nutritional value and bioavailability. At the physicochemical level, enzymatic hydrolysis breaks down tightly folded protein molecules into multiple peptide segments by cleaving peptide bonds. This cleavage inevitably leads to two physical consequences: firstly, it exposes hydrophobic amino acid residues previously encapsulated within the protein to the aqueous environment; secondly, each cleavage of a peptide bond generates a new reactive amino group at the end of the newly formed peptide segment. These two physical consequences directly lead to a technical problem in existing processes: under high-concentration or long-term industrial enzymatic hydrolysis conditions, a large number of newly exposed hydrophobic regions attract each other due to hydrophobic interactions, causing peptide aggregation and precipitation, thus affecting the product's solubility and stability. Furthermore, certain specific sequences of hydrophobic peptides are also major sources of bitterness. To address this, the industry practice is to perform post-treatment of the aggregates and bitter substances after the enzymatic hydrolysis reaction by adding independent downstream processing steps such as activated carbon adsorption, flavor masking, or high-pressure homogenization.

[0003] To simplify the process, there are some ideas for improvement from the source, but their application is limited. For example, screening or modifying a protease that can avoid the formation of hydrophobic bitter peptides is difficult in terms of technical feasibility and economics due to the diversity of protein substrate sources and structures. Another idea is to optimize the downstream adsorption or masking process, but this will not only increase additional production costs and energy consumption, but may also cause the simultaneous loss of nutrient peptides when removing target impurities, and make the product formulation more complicated.

[0004] It is evident that existing technologies are based on a default premise: the enzymatic hydrolysis reaction and product quality degradation occur simultaneously, while quality control is achieved through subsequent remedial steps. This confines the entire protein enzymatic modification process to a framework of degradation followed by remediation. Specifically, existing technologies suffer from the following shortcomings: 1. The process units are independent, with protein degradation and product quality control processes separated, resulting in a long production process and numerous control steps; 2. Key product quality indicators, such as solubility and flavor, cannot be actively controlled online during the enzymatic hydrolysis reaction, but can only passively accept the initial state of the hydrolysate, increasing the difficulty of controlling batch-to-batch consistency of the final product; 3. The enzymatic hydrolysis process is treated as a single degradation step, and the accompanying changes in protein molecular structure and chemical endpoints are not utilized as a technical resource for simultaneously achieving functional modification of the product. Therefore, how to establish a new process that allows the enzymatic degradation of proteins and the functional modification of peptides to occur simultaneously within the same reaction unit, thereby avoiding quality degradation caused by the exposure of hydrophobic peptides, is the technical problem this invention aims to solve. Summary of the Invention

[0005] This invention provides an enzymatic modification of exogenous proteins and its application in baked goods. Its main purpose is to solve the problem that in existing protein enzymatic hydrolysis processes, the degradation process is separated from the quality control process, and the quality problems that have already occurred can only be dealt with by complex downstream processes.

[0006] To achieve the above objectives, the present invention provides an enzymatic modification of exogenous proteins, which includes the following method steps:

[0007] Step a: In an aqueous medium, exogenous protein, proteolytic enzyme, a reducing sugar, and a food-grade non-reducing polysaccharide are mixed before the enzymatic hydrolysis reaction begins to form an initial reaction solution, wherein the initial mass ratio of the reducing sugar to the exogenous protein is set to 5% to 20%.

[0008] Step b, at 30 Up to 60 Under specific temperature conditions, proteolytic enzymes initiate the enzymatic hydrolysis of exogenous proteins.

[0009] In step c, during the enzymatic hydrolysis reaction in step b, the α-amino group generated at the N-terminus of the newly formed peptide segment by the cleavage of peptide bonds by the proteolytic enzyme covalently bonds with the carbonyl group of the reducing sugar already present in the initial reaction solution. In step a, the food-grade non-reducing polysaccharide forms a micro-network structure in the initial reaction solution that can delay the interdiffusion of hydrophobic peptide molecules generated by the enzymatic hydrolysis reaction in step b. Furthermore, the settings of steps a and b ensure that the rate of the chemical reaction of covalent bonding that occurs in step c is always higher than the rate of physical aggregation of hydrophobic peptide molecules due to interdiffusion throughout the entire enzymatic hydrolysis reaction.

[0010] Preferably, the enzymatic hydrolysis reaction is initiated in the initial reaction solution at a preset pH value where the proteolytic enzyme can operate; and the initial reaction solution also contains one or more catalysts selected from phosphates and citrates to increase the reaction rate of covalent bonding between the α-amino group generated at the N-terminus of the nascent peptide and the carbonyl group of the reducing sugar under the preset pH value conditions.

[0011] Preferably, the reducing sugar is one or more reducing oligosaccharides that have prebiotic functions.

[0012] Preferably, the reducing oligosaccharide with prebiotic function is selected from one or more of fructooligosaccharides, galactooligosaccharides and isomaltooligosaccharides.

[0013] Preferably, the food-grade non-reducing polysaccharide is one or more food-grade colloids selected from xanthan gum, guar gum, and high molecular weight pectin, and the initial reaction solution also contains a substance to inhibit the reduction of sugars at 30°C. Up to 60 Food-grade stabilizers that undergo chemical degradation under certain temperature conditions.

[0014] Preferably, the food-grade stability protectant is a sulfite.

[0015] Preferably, the initial reaction solution also contains one or more polyols selected from glycerol, sorbitol and propylene glycol, which are used to reduce the water activity of the aqueous medium.

[0016] Preferably, the relationship between the chemical reaction rate and the physical aggregation rate of covalent bonding is quantitatively characterized by the following rule: at any time point during the enzymatic hydrolysis reaction, the reaction rate is anchored. With aggregation reaction rate In between, continuously satisfy The relationship; in which, the anchoring reaction rate The consumption rate of free amino groups and the aggregation reaction rate are determined by the phthalaldehyde method. The rate of increase in light scattering intensity is measured at a detection wavelength set within the wavelength range of 400 nm to 800 nm.

[0017] Preferably, the exogenous protein is one or more proteins derived from soybean protein, pea protein, whey protein, and casein; and the proteolytic enzyme is one or more proteases adapted to the exogenous protein.

[0018] The application of exogenous protein hydrolysates in the preparation of baked goods includes adding protein hydrolysates as nutritional fortifiers and dough improvers to baked goods ingredients containing flour.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] 1. By pre-setting exogenous protein protease and one or more food-grade reducing sugars in the same aqueous medium before the enzymatic hydrolysis reaction begins, each cleavage action of the protease on the peptide bond instantly generates a new reactive amino group at the N-terminus of the generated peptide. This amino group immediately covalently bonds with the reducing sugar already present in the medium. This integrated coupling of protein degradation and peptide functionalization in time and space avoids the traditional process of first hydrolyzing to generate a large number of hydrophobic peptides and then treating their aggregation and bitterness in subsequent steps, thus avoiding the molecular basis for the generation of undesirable properties.

[0021] 2. The initial composition includes a food-grade, non-reducing, high-molecular-weight polysaccharide as a kinetic regulator. This polysaccharide pre-forms a three-dimensional network structure in an aqueous medium that can slow down the Brownian motion of molecules. When the enzymatic hydrolysis reaction begins, this network structure physically inhibits the diffusion and collision of newly generated hydrophobic peptide molecules. This provides a time window for the covalent bonding reaction between the N-terminal amino group and the reducing sugar, unaffected by the physical process of peptide aggregation. This ensures that the chemical modification reaction maintains its kinetic advantage over the physical aggregation process even under high substrate concentrations in industrial production conditions, thereby improving the stability and applicability of the entire process under different production concentration requirements.

[0022] 3. This invention limits the reducing sugar to one or more reducing oligosaccharides that inherently possess prebiotic functions, such as fructooligosaccharides or galactooligosaccharides. Under this setting, the same technical action of enzymatically cleaving peptide bonds and triggering covalent bonding not only endows the nascent peptide with hydrophilic properties but also covalently links a functional molecule (prebiotic) with specific biological activity to the peptide carrier. This results in the final protein hydrolysate no longer being a simple physical mixture of nutrients and prebiotics, but rather a novel chemical entity that integrates two functional attributes at the molecular level: an easily absorbed nitrogen source and a targeted biological activity signal. This provides a more clearly structured and functionally integrated base material for the development of functional foods. Attached Figure Description

[0023] Figure 1 This is a schematic diagram illustrating the technical principle of inhibiting hydrophobic aggregation in the integrated enzymatic hydrolysis modification of this invention.

[0024] Figure 2 This is a schematic diagram of the protease activity calibration based on the reaction process curve of the present invention;

[0025] Figure 3 This is a schematic diagram illustrating the prior art of degradation followed by treatment process and its defects. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] This invention discloses an enzymatic modification of exogenous proteins and its application in baked goods. The technical solution involves constructing an initial reaction solution and initiating a reaction process triggered by a proteolytic enzyme, where protein degradation and peptide functionalization occur simultaneously within the physicochemical environment defined by this solution. This process utilizes the α-amino group generated at the N-terminus of the newly formed peptide by the proteolytic enzyme's cleavage of peptide bonds to covalently link with the carbonyl group of a reducing sugar pre-existing in the initial reaction solution. Through synergistic regulation of the chemical reaction rate and physical diffusion rate within the system, the rate of the covalently linked chemical reaction remains consistently higher than the physical aggregation rate caused by the interdiffusion of hydrophobic peptide molecules throughout the enzymatic hydrolysis reaction. This yields a functional protein hydrolysate applicable to the baked goods industry. In a specific application, for example, it can be used to develop a high-solubility, non-toxic, and non-toxic protein hydrolysate for high-protein baked goods. When hydrolyzing soy protein for bitterness and dough improvement, conventional enzymatic hydrolysis methods can lead to product aggregation and bitterness due to the exposure of hydrophobic peptides, limiting its addition in baking formulations. To address this issue, the enzymatic modification method of this invention involves pre-mixing an exogenous protein (e.g., 100 parts by weight of soy protein isolate) as a substrate, a proteolytic enzyme (e.g., 1 part by weight of a complex protease) as a reaction trigger and site-forming tool, a reducing sugar (e.g., 10 parts by weight of isomaltooligosaccharide) as a covalent anchoring agent, and a food-grade non-reducing polysaccharide (e.g., 0.5 parts by weight of xanthan gum) as a kinetic regulator to form an initial reaction solution. By integrating all functional components before the reaction is initiated, protein degradation is transformed into a process that occurs simultaneously with peptide hydrophilic modification, thereby inhibiting product aggregation and the formation of bitter substances.

[0028] Furthermore, under high-concentration industrial production conditions, the physical aggregation rate of newly generated hydrophobic peptides increases due to the reduced intermolecular distance, affecting the efficiency of the covalent anchoring reaction. Therefore, the food-grade non-reducing polysaccharide xanthan gum contained in the initial reaction solution forms a three-dimensional network structure in the aqueous medium. This network structure, by increasing the microviscosity of the medium, physically hinders the Brownian motion and interdiffusion of newly generated hydrophobic peptide molecules during enzymatic hydrolysis, providing the necessary reaction time for the chemical reaction of covalent linkage between the α-amino group at the N-terminus of the nascent peptide and the reducing sugar. To ensure this kinetic advantage, the system calibrates the chemical reaction rate and physical aggregation rate of covalent linkage through a quantitative relationship; that is, at any time point during the enzymatic hydrolysis reaction, the anchoring reaction rate and the aggregation reaction rate continuously satisfy the following relationship. ;in, To anchor the reaction rate, its unit is mol / (L·s), which was determined by a procedure based on the o-phthalaldehyde (OPA) method for measuring the consumption rate of free amino groups. The aggregation reaction rate, expressed in I / (s), was determined using a rate-increasing assay based on light scattering intensity. By introducing a kinetic modifier and adhering to this quantitative relationship, the chemical anchoring modification rate was consistently higher than the physical aggregation rate, improving the stability of the process under different production concentration requirements. Furthermore, the optimal reaction pH for different proteolytic enzymes is related to their catalytic activity, but this pH may not be favorable for the carbonyl-amine condensation reaction, which forms the basis of the anchoring reaction. This constitutes a constraint on the kinetic environment of the multiple reaction system. To overcome this constraint, the present invention further includes a... Catalysts, such as food-grade phosphates or citrates, are used to target and enhance the reaction rate between nascent N-terminal α-amino groups and reducing sugar carbonyl groups in neutral or slightly acidic pH processes, which are required by specific proteases but are unfavorable to the rate of covalent linkage reactions, such as at pH 6.5. In this way, the global pH of the process can be set according to the highest activity of the selected protease without compromising the efficiency of the anchoring reaction. This design decouples the optimal pH conditions of the enzymatic reaction from the kinetic requirements of the covalent linkage reaction, expanding the application range of the technical solution.

[0029] In a preferred embodiment, the selection of the covalent anchoring agent is functionally limited, namely, the reducing sugar is one or more reducing oligosaccharides with prebiotic functions, such as one or more of fructooligosaccharides, galactooligosaccharides, and isomaltooligosaccharides. Under this setting, the same technical action of enzymatic hydrolysis cleaving peptide bonds and triggering covalent bonding not only connects a hydrophilic group to the nascent peptide segment but also covalently links a prebiotic molecule with specific biological activity to the peptide segment carrier, making the final protein hydrolysate a chemical entity integrating easily absorbed nitrogen source and prebiotic functional properties, providing a more functionally integrated base material for the development of functional baked goods. To ensure the robustness of the process, the scheme also configures two technical steps that affect the certainty of the reaction endpoint, one of which is to handle the reducing sugar under mild heating enzymatic hydrolysis conditions (e.g., at 30°C). Up to 60 To address potential chemical degradation issues within the given temperature range, the initial reaction solution is formulated to also contain a food-grade stabilizer, such as a sulfite, which inhibits the non-specific chemical degradation of reducing sugars under enzymatic hydrolysis conditions, thus ensuring the effective concentration of the covalent anchoring agent throughout the reaction cycle. Secondly, the microenvironment of the reaction medium is constructed by including one or more polyols selected from glycerol, sorbitol, and propylene glycol in the initial reaction solution to reduce the water activity of the aqueous phase. In an environment with reduced water activity, the carbonyl-amine condensation reaction, as a dehydration reaction, is favored. The process can also inhibit the tendency of hydrophobic aggregation to a certain extent, thereby enhancing the advantages of anchoring reaction over aggregation reaction; finally, the protein hydrolysate obtained by any of the aforementioned methods can be used in the preparation of baked goods, including adding the protein hydrolysate as a nutrient fortifier and dough improver to baked goods ingredients containing flour to prepare baked goods; since the molecular structure of the product contains covalently linked glycosyl groups (hydrophilic ends) and peptide backbones (hydrophobic ends) and has amphiphilicity, it can function as an emulsifier in the dough system, which helps to improve the gluten network structure and the texture of the final product.

[0030] Example 1: In an industrial production process for preparing a high-concentration pea protein nutrient base, the process objective is to prepare a pea protein hydrolysate with a final protein concentration of not less than 20% (w / v) in a 5000-liter reactor. The hydrolysate must have a solubility of not less than 95% and be free of unpleasant flavor for subsequent liquid nutrient formulation. When using a conventional enzymatic hydrolysis method, i.e., pea protein isolate and proteolytic enzymes are reacted in an aqueous medium at 50°C... During the reaction, after 30 minutes, turbidity and flocculent precipitate appeared in the liquid inside the reactor, indicating that the hydrophobic peptides exposed by enzymatic hydrolysis had physically aggregated under high substrate concentrations. To address this issue, an enzymatic modification method for exogenous proteins according to this invention is employed. The procedure involves first constructing an initial reaction solution containing all functional components in an aqueous medium within a 5000-liter reactor. This solution includes 1000 kg of pea protein isolate, 100 kg of fructooligosaccharides, 5 kg of guar gum, and 2 kg of trisodium citrate. After all components are evenly dispersed, a proteolytic enzyme adapted to pea protein is added, and the pH of the reaction system is set to a suitable level of 6.8 for the protease. The reaction is then initiated at 50°C. Enzymatic hydrolysis reaction at temperature.

[0031] After the reaction begins, the cleavage of peptide bonds by the proteolytic enzymes simultaneously exposes the hydrophobic regions of the peptides and generates N-terminal α-amino groups. At this point, two pre-defined functional components in the initial reaction solution begin to function. First, guar gum, acting as a kinetic regulator, forms a micro-network structure in the aqueous medium that physically hinders the interdiffusion of newly generated hydrophobic peptide molecules, thus slowing down the physical aggregation rate. This provides time for the chemical reaction to proceed; correspondingly, the fructooligosaccharide, acting as a covalent anchoring agent, utilizes this time to covalently link with the N-terminal α-amino group of the nascent peptide, thus increasing the rate of this anchoring reaction. Due to the catalytic effect of trisodium citrate, it can be maintained at a high level under pH 6.8 conditions, thereby enabling... Higher than that delayed by physical obstruction This method synchronizes the enzymatic hydrolysis process with the product quality control process by kinetic management of the chemical anchoring reaction and the physical aggregation process. After 4 hours of enzymatic hydrolysis, a sample is taken from the reactor. The resulting protein hydrolysate is a homogeneous liquid without visible precipitate. After centrifugation and drying, its solubility in water is measured to be 98%. At the same time, the sensory evaluation team determined that the hydrolysate has no obvious bitterness. This product can be used as a high-concentration protein nutrient base for the subsequent production of liquid nutrients without the need for post-treatment steps such as activated carbon debittering or high-pressure homogenization.

[0032] Example 2: To verify the enzymatic modification method of exogenous proteins of the present invention and to determine the range of addition amounts of the key component, reducing sugar, this comparative experiment was set up; the experimental platform was a machine with jacketed heating and temperature control function, and the temperature control accuracy was ±0.1. A 1-liter glass reactor was used, equipped with an adjustable-speed stirrer. Product performance testing equipment included a UV-Vis spectrophotometer for determining the nitrogen solubility index to characterize solubility, and a panel of five trained sensory evaluators who used a 5-point intensity scale to evaluate bitterness, where 1 point represents no bitterness and 5 points represent extremely strong bitterness. The experiment consisted of five sample groups: one experimental group and four control groups. All sample groups used the same whey protein isolate as the exogenous protein substrate. The initial protein concentration in the aqueous medium was 15% (w / v), the amount of proteolytic enzyme added was 1% of the protein mass, and the reaction temperature was 55.0°C. The reaction pH was 7.0, and the total reaction time was 4 hours. The formulation differences of each group were set as follows: the experimental group contained 15% by mass of reducing sugar (maltose) and 0.5% by mass of non-reducing polysaccharide (xanthan gum) in its initial reaction solution; control group 1 contained no non-reducing polysaccharide in its initial reaction solution; control group 2 contained no reducing sugar in its initial reaction solution; control group 3 contained 2% reducing sugar in its initial reaction solution; and control group 4 contained 25% reducing sugar in its initial reaction solution. After the experiment started, about 1 hour into the reaction, white flocculent matter appeared in the reaction solutions of control group 1 and control group 2 through the visual window of the reactor, and gradually increased in subsequent reactions. However, the solutions of the experimental group, control group 3, and control group 4 remained homogeneous throughout the 4-hour reaction. After the enzymatic hydrolysis reaction of all groups was completed and the proteolytic enzymes were inactivated by heating, the performance of the protein hydrolysates obtained from each group was tested. The comparison data of the key indicators are shown in Table 1.

[0033] Table 1: Performance comparison of protein hydrolysates from different groups.

[0034] Sample group name Reducing sugar addition (%) Non-reducing polysaccharides Product Appearance Nitrogen solubility index (%) Bitterness rating experimental group 15.0 Add to Clarity and transparency 97.5 1.4 Control group 1 15.0 No additions Slightly cloudy 72.3 3.1 Control group 2 0 Add to Turbid, with sediment 48.6 4.5 Control group 3 2.0 Add to Clarity and transparency 81.4 3.6 Control group 4 25.0 Add to Clear, slightly yellow 96.8 1.6

[0035] Table 1 shows that the nitrogen solubility index of the product in the experimental group was 97.5%, and the bitterness score was 1.4. Control group 2, which did not contain any reducing sugar, had a nitrogen solubility index of only 48.6% and a bitterness score as high as 4.5. Compared to the experimental group, control group 1, lacking non-reducing polysaccharides as kinetic regulators, had a nitrogen solubility index of 72.3% and a bitterness score of 3.1. Data from control group 3 shows that when the amount of reducing sugar added as a covalent anchor was 2%, the nitrogen solubility index of the product was 81. The product of control group 4 was close to that of the experimental group in terms of nitrogen solubility index and bitterness score, but its solution showed a slightly yellow color due to chemical side reactions in the later stage of the reaction. The experimental results showed that, under the experimental conditions, the experimental group, which contained reducing sugars and non-reducing polysaccharides and whose reducing sugar content was in the range of 5% to 20%, produced protein hydrolysates with measurable differences in solubility and flavor indicators compared with the control group, which lacked key components or whose key component content was outside this range.

[0036] To further verify, from the perspective of kinetic regulation, the indispensability of non-reducing polysaccharides as kinetic regulators in inhibiting the physical aggregation of hydrophobic peptides, the following comparative example 1 was set up.

[0037] Comparative Example 1: This comparative example aims to verify that even in the presence of sufficient reducing sugars as covalent anchors, the conventional enzymatic hydrolysis process cannot effectively inhibit the physical aggregation of hydrophobic peptides in the absence of non-reducing polysaccharides as kinetic regulators, thus leading to product quality degradation. The experimental conditions were basically the same as those in Example 2, except that no non-reducing polysaccharides were added to the initial reaction solution in this comparative example (i.e., the amount of xanthan gum added was 0). Specifically, in a 1-liter glass reactor with a jacketed heating and temperature control function, whey protein isolate was used as an exogenous protein substrate, and its initial concentration in the aqueous medium reached 15% (w / v). Subsequently, reducing sugar (maltose) accounting for 15% of the protein mass was added. After mixing evenly, the pH of the reaction system was adjusted to 7.0, and the temperature was precisely controlled at 55.0°C. Finally, a proteolytic enzyme at 1% of the protein mass was added, and the enzymatic hydrolysis reaction was initiated for a total duration of 4 hours. After the reaction started, the solution state was continuously monitored through the visual window of the reactor. At approximately 55 minutes, a visible milky-white turbidity began to appear in the initially clear solution. At approximately 70 minutes, the turbidity intensified, and fine white flocculent matter began to form. After the 4-hour reaction, the liquid in the reactor exhibited a non-uniform turbid state, with a small amount of identifiable precipitate at the bottom. The final enzymatic hydrolysis product was subjected to the same heat inactivation treatment, and its key performance indicators were tested. The results are summarized in Table 2.

[0038] Table 2: Performance comparison of the products of Example 2 and Comparative Example 1.

[0039] Sample group name Reducing sugar addition (%) Non-reducing polysaccharide addition (%) Key process phenomenon (when the reaction proceeds to 70 minutes) Appearance of the final product Nitrogen solubility index (%) Bitterness rating Example 2 Test Group 15.0 0.5 The solution remains homogeneous and clear. Clarity and transparency 97.5 1.4 Comparative Example 1 15.0 0 The solution became cloudy and formed white flocculent matter. Slightly cloudy with sediment 72.3 3.1

[0040] Table 2 objectively shows that, compared with the experimental group of Example 2, Comparative Example 1, which only lacked non-reducing polysaccharides, experienced a sharp decrease in the nitrogen solubility index of its product from 97.5% to 72.3%, while the bitterness score significantly increased from 1.4 to 3.1. The process record further confirms that the quality deterioration and the observable physical aggregation phenomena (turbidity and flocculation) during the reaction were synchronous in time. This result indicates that, without the physical hindrance of a micro-network constructed by non-reducing polysaccharides, even with the presence of reducing sugars in the system, the physical aggregation rate of hydrophobic peptides (…) The rate of covalent anchoring of nascent amino acids and sugars remains significantly higher than that of nascent amino acids and sugars. This makes it impossible to prevent the formation of aggregates and bitter peptides from the source. This directly confirms the necessity of the technical concept of this invention, which ensures that the chemical anchoring rate is always higher than the physical aggregation rate through kinetic regulation.

[0041] Example 3: This example combines Figures 1 to 3The enzymatic modification of exogenous proteins and its application in baked goods are explained, such as... Figure 1 As shown, in this reaction system, exogenous protein serves as the protein substrate, proteolytic enzymes act as enzyme catalysts, reducing sugars act as covalent anchors, and non-reducing polysaccharides provide kinetic regulation. These four components participate in an integrated enzymatic hydrolysis and modification reaction. In this reaction, the hydrophobic peptides and newly formed N-terminal amino groups generated during the enzymatic hydrolysis process are guided to two parallel pathways. The newly formed N-terminal amino groups are directly converted into the target product, functional protein hydrolysate, through covalent anchoring modification. Meanwhile, the physical aggregation tendency of the hydrophobic peptides is inhibited by the physical hindrance provided by the non-reducing polysaccharides, thereby preventing the formation of aggregates and bitter peptides at the source.

[0042] like Figure 2 As shown, the vertical axis represents the free amino group concentration in mmol / L, and the horizontal axis represents the reaction time in min. The figure shows a solid line representing the baseline batch, a dashed line representing the high activity of the new batch A, and a dotted line representing the low activity of the new batch B. By monitoring the generation rate of free amino groups in the standard reaction system of different batches of enzyme preparations, the differences in their activity can be quantitatively assessed. Based on this, the total reaction time in the production process can be adjusted, thereby eliminating the influence of the differences between raw material batches on the final degree of hydrolysis of the product.

[0043] like Figure 3 As shown, protein raw materials and proteolytic enzymes initiate an enzymatic hydrolysis reaction in the reaction system. After the peptide bonds break, hydrophobic peptides are immediately generated, which then lead to peptide aggregation and the formation of bitter substances, resulting in quality problems such as decreased product solubility. To solve this problem, the turbid enzymatic hydrolysate needs to be transferred to a separate post-processing step. The product can be salvaged by activated carbon adsorption, high-pressure homogenization, or flavor masking. The final post-processed product not only faces the problems of nutrient loss and increased cost, but also reflects the technical limitations of separating the protein degradation and quality control processes.

[0044] Example 4: To determine the anchoring reaction rate in a specific protein and proteolytic enzyme system. Relative to the aggregation reaction rate To determine the minimum kinetic advantage ratio and obtain a qualified final product, the following standardized engineering calibration procedure can be adopted. In the development of a process for enzymatic modification of casein as an exogenous protein, it is necessary to calibrate a kinetic control threshold for the raw material system that ensures the solubility of the final product is not less than 95%. The initial experimental conditions are as follows: in a 1-liter isothermal reactor equipped with online dynamic light scattering monitoring and automatic sampling functions, an initial casein reaction solution with a protein concentration of 15% (w / v) is prepared, containing 12% maltose as a covalent anchoring agent, and a suitable protease is added. The reaction temperature is maintained at 50.0°C. The pH value was 7.0. The calibration procedure involved setting up a series of parallel experimental groups. The only variable between the groups was the concentration of xanthan gum, which served as a kinetic regulator. The concentration gradient was set from 0% to 0.8% (w / v) with a step size of 0.1%. For each group, after the enzymatic hydrolysis reaction was started, the rate of increase in the light scattering intensity of the reaction solution was recorded in real time using a dynamic light scattering monitor. This rate was used as the aggregation reaction rate of that group. Meanwhile, samples were taken every 15 minutes using an automated sampling system, and the consumption rate of free amino groups in the samples was determined using the o-phthalaldehyde method. This rate was used as the anchoring reaction rate for that sample group. Four hours after the reaction, the final product was collected and its nitrogen solubility index was determined by centrifugation.

[0045] The measurements of each sample group during the reaction period and The average ratio was correlated with the nitrogen solubility index of the final product of the sample group. The results showed that when When the ratio is below 1.1, the nitrogen solubility index of the product increases rapidly with increasing ratio; when the ratio is between 1.1 and 1.3, the increase in nitrogen solubility index tends to level off and stabilizes above 95%; however, when the ratio exceeds 1.3, further increasing the xanthan gum concentration no longer has a significant effect on increasing the nitrogen solubility index. Based on this data, and considering the fluctuations in industrial production and setting a safety margin, the kinetic advantage ratio that should be satisfied by the process operation in this casein proteolytic system was determined to be: .

[0046] Example 5: In an industrial production process, to address potential activity differences between different batches of proteolytic enzymes, a standardized raw material batch pre-inspection and process parameter fine-tuning procedure is implemented before a new batch of enzyme preparation is introduced into large-scale production. This procedure is carried out in a 1-liter standardized laboratory reaction system. The material formulation, reaction temperature, and pH settings of this system are consistent with the experimental group settings used to prepare qualified products in the previous examples, and it is equipped with timed automatic sampling equipment and analytical instruments for performing the phthalaldehyde method. The pre-inspection procedure involves adding the new proteolytic enzyme batch to be tested into the standardized... The enzymatic hydrolysis reaction was initiated in a laboratory reaction system. After the reaction started, an automated sampling device extracted samples from the reaction system every 20 minutes, and the concentration of free amino groups in the samples was measured by an analytical instrument. The continuously measured free amino group concentration data were plotted as a reaction progress curve over time, and the real-time increase rate of free amino group concentration was calculated based on the curve. The procedure defines the endpoint of the enzymatic hydrolysis reaction as the moment when the increase rate of free amino group concentration first drops to below 5% of its initial maximum rate, and records the total time required to reach the endpoint and the total concentration of free amino groups at the endpoint, which are used as indicators of the activity of the proteolytic enzyme in the batch to be tested.

[0047] After obtaining the activity indicators of a specific batch of proteolytic enzymes, these indicators are used as the basis for adjusting the total enzymatic hydrolysis reaction time under large-scale industrial production. If the time required for a baseline batch of enzyme preparation to reach the reaction endpoint is 4 hours, and a new batch of enzyme preparation, after being tested according to this procedure, requires 3.5 hours to reach the same reaction endpoint, then when using this new batch of enzyme preparation for large-scale production, the total enzymatic hydrolysis reaction time in its process procedure is adjusted accordingly to 3.5 hours. In this way, the influence of batch-to-batch differences in raw materials on the final degree of hydrolysis of the product can be eliminated.

[0048] Example 6: When applying the enzymatic modification method of the present invention to a baking-specific soy protein isolate from a specific source, the following offline optimization parameter-finding procedure needs to be executed to determine the combination of process parameters that simultaneously achieves the highest solubility and the lowest bitterness value. The objective function of this procedure is to find a set of reaction temperature and pH values ​​that maximize the nitrogen solubility index of the product and minimize the bitterness score. The fixed parameters of the experiment are: protein concentration 15% (w / v), maltose addition 15%, xanthan gum addition 0.5%, and reaction time calibrated according to the above method. This parameter-finding procedure uses response surface methodology and is executed in a central composite design experiment consisting of 13 experimental groups. The variable parameters of the experiment are reaction temperature and reaction pH values, the range of which is determined based on previous single-factor experiments. The five levels of reaction temperature are set to 45.0, 47.5, 50.0, 52.5, and 55.0. The five pH levels for the reaction were set to 6.5, 6.8, 7.1, 7.4, and 7.7. Each experimental sample group was subjected to enzymatic hydrolysis in a 1-liter standardized reactor under a specific temperature and pH combination determined by the above experimental design.

[0049] After the reactions of all 13 experimental groups were completed, the nitrogen solubility index and bitterness score of the final product of each group were measured, and these two indicators were used as response values. The experimental data were fitted with quadratic polynomial regression to establish mathematical models for the nitrogen solubility index and bitterness score with respect to reaction temperature and pH. Contour plots of the two response values ​​were obtained by analyzing the models, and the two plots were overlaid to find the optimal parameter region. The analysis results showed that there exists a parameter combination region where the nitrogen solubility index can be maintained above 96% and the bitterness score can be maintained below 1.5. The center point of this region is the optimal combination of process parameters for this specific soybean protein raw material system, which was determined to be a reaction temperature of 51.5°C. The reaction pH was 7.2.

[0050] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. An enzymatic modification of an exogenous protein, characterized in that, Enzymatic modification includes the following steps: Step a: In an aqueous medium, exogenous protein, proteolytic enzyme, a reducing sugar, and a food-grade non-reducing polysaccharide are mixed before the enzymatic hydrolysis reaction begins to form an initial reaction solution, wherein the initial mass ratio of the reducing sugar to the exogenous protein is set to 5% to 20%. Step b, at 30 Up to 60 Under certain temperature conditions, proteolytic enzymes initiate the enzymatic hydrolysis of exogenous proteins; In step c, during the enzymatic hydrolysis reaction in step b, the α-amino group generated at the N-terminus of the newly formed peptide segment by the cleavage of peptide bonds by the proteolytic enzyme covalently bonds with the carbonyl group of the reducing sugar already present in the initial reaction solution. In step a, the food-grade non-reducing polysaccharide forms a micro-network structure in the initial reaction solution that can delay the interdiffusion of hydrophobic peptide molecules generated by the enzymatic hydrolysis reaction in step b. Furthermore, the settings of steps a and b ensure that the rate of the chemical reaction of covalent bonding that occurs in step c is always higher than the rate of physical aggregation of hydrophobic peptide molecules due to interdiffusion throughout the entire enzymatic hydrolysis reaction.

2. The enzymatic modification of exogenous proteins according to claim 1, characterized in that, In the initial reaction solution, the enzymatic hydrolysis reaction is initiated at a preset pH value that allows the proteolytic enzyme to function; and the initial reaction solution also contains one or more catalysts selected from phosphates and citrates to enhance the reaction rate of covalent bonding between the α-amino group generated at the N-terminus of the nascent peptide and the carbonyl group of the reducing sugar under the preset pH value.

3. The enzymatic modification of exogenous proteins according to claim 1, characterized in that, Reducing sugars are one or more reducing oligosaccharides that have prebiotic functions.

4. The enzymatic modification of exogenous proteins according to claim 3, characterized in that, The reducing oligosaccharides with prebiotic functions are selected from one or more of fructooligosaccharides, galactooligosaccharides, and isomaltooligosaccharides.

5. The enzymatic modification of exogenous proteins according to claim 1, characterized in that, Food-grade non-reducing polysaccharides are one or more food-grade colloids selected from xanthan gum, guar gum, and high-molecular-weight pectin. The initial reaction solution also contains a substance to inhibit the reduction of sugars at 30°C. Up to 60 Food-grade stabilizers that undergo chemical degradation under certain temperature conditions.

6. The enzymatic modification of exogenous proteins according to claim 5, characterized in that, The food-grade stabilizer is a sulfite.

7. The enzymatic modification of exogenous proteins according to claim 1, characterized in that, The initial reaction solution also contains one or more polyols selected from glycerol, sorbitol and propylene glycol, which are used to reduce the water activity of the aqueous medium.

8. The enzymatic modification of exogenous proteins according to claim 1, characterized in that, The relationship between the chemical reaction rate and the physical aggregation rate of covalent bonding is quantitatively characterized by the following rule: at any time point during the enzymatic hydrolysis reaction, the anchoring reaction rate... With aggregation reaction rate In between, continuously satisfy The relationship; in which, the anchoring reaction rate The consumption rate of free amino groups and the aggregation reaction rate are determined by the phthalaldehyde method. The rate of increase in light scattering intensity is measured at a detection wavelength set within the wavelength range of 400 nm to 800 nm.

9. The enzymatic modification of exogenous proteins according to claim 1, characterized in that, The exogenous protein is one or more proteins derived from soy protein, pea protein, whey protein, and casein; the proteolytic enzyme is one or more proteases adapted to the exogenous protein.

10. The application of the exogenous protein hydrolysate obtained by any one of claims 1 to 9 in the preparation of baked goods, characterized in that, Applications include using protein hydrolysates as nutritional fortifiers and dough improvers, adding them to baking ingredients containing flour to prepare baked goods.

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