Method for improving functional characteristics of casein

Abstract

The invention belongs to the technical field of food processing and functional dairy product development, and particularly relates to a method for improving casein functional characteristics, which comprises the following steps: S1, dissolving casein in a buffer solution to obtain a casein solution; s2, performing cold plasma treatment on the casein solution by using inert gas as working gas, uniformly stirring after the cold plasma treatment is completed, and standing and incubating to obtain a pretreated casein solution; s3, adding oligosaccharide into the pretreated casein solution, uniformly mixing, standing, and freeze-drying to obtain a mixture freeze-dried powder; and S4, carrying out glycosylation reaction on the freeze-dried powder of the mixture to improve the functional characteristics of casein. Based on the combination of plasma pretreatment and a glycosylation technology, functional modification of casein is realized. According to the method disclosed by the invention, the IgE binding capacity of the casein is effectively reduced, the allergy risk is reduced, and the solubility, emulsibility, foamability and thermal stability of the casein are also remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of food processing and functional dairy product development technology, specifically relating to a method for improving the functional properties of casein. Background Technology

[0002] Milk is an indispensable part of the human diet, highly valued for its excellent nutritional value and multiple functions in the food system. Casein, as the main protein component of milk, accounts for about 80% of the total protein content. Due to its affordability, ease of production, and excellent nutritional and processing characteristics, it plays a central role in the dairy and food industries. However, under complex processing conditions such as high temperature and acid / alkali conditions, the solubility, emulsifying properties, foaming properties, and stability of casein are limited. Furthermore, because casein molecules carry multiple antigenic epitopes that can be recognized by immunoglobulin E, they are prone to triggering allergic immune reactions. These issues limit its further widespread application in dairy product development.

[0003] To improve the function and safety of casein, various modification methods have been explored in existing technologies, including heat treatment, enzymatic hydrolysis, and Maillard reaction. However, these methods all have significant limitations. The high temperatures of heat treatment easily lead to irreversible denaturation and aggregation of proteins, destroying their natural structure and functional properties, and have limited effect on reducing allergenicity. While enzymatic hydrolysis can reduce allergenicity, it easily produces bitter peptides, affecting product flavor, and the degree of hydrolysis is difficult to control precisely, often resulting in functional loss. The Maillard reaction, as a relatively green modification method, can improve protein function to some extent and mask some allergen epitopes. However, existing Maillard reaction technologies have the following drawbacks: they typically rely on high temperatures above 80°C and long processing times, easily causing protein denaturation, aggregation, and stability problems; secondly, the Maillard reaction has low modification efficiency, with limited improvement in allergenicity. Furthermore, recent studies have attempted to use cold plasma technology for protein modification, but existing cold plasma treatments may generate reactive nitrogen species that can cause excessive protein oxidation and uncontrollable modification, making it difficult to achieve a synergistic improvement in function and safety.

[0004] Therefore, how to achieve efficient and controllable glycosylation modification of casein under mild conditions, while significantly improving its functional properties and reducing its allergenicity, has become a technical bottleneck that urgently needs to be overcome in the field of casein modification. Summary of the Invention

[0005] This invention provides a method for improving the functional properties of casein, which not only effectively reduces the IgE binding capacity of casein and reduces the risk of allergies, but also significantly improves its solubility, emulsification, foaming properties and thermal stability.

[0006] It solves the problems existing in the current technology.

[0007] The technical solution adopted in this invention is: This invention provides a method for improving the functional properties of casein, comprising the following steps: S1: Dissolve casein in a buffer solution at pH 7.4 to obtain a casein solution; S2: Using an inert gas as the working gas, the casein solution is subjected to cold plasma treatment. After the treatment is completed, the solution is stirred evenly and allowed to stand for incubation to obtain a pretreated casein solution. S3: Add oligosaccharides to the pretreated casein solution, mix well, let stand, freeze dry, and obtain the freeze-dried powder of the mixture; the dry mass ratio of oligosaccharides to casein is 1:1~2; S4: Glycosylation of the lyophilized mixture powder can improve the allergenicity, solubility, emulsification, foaming properties and thermal stability of casein.

[0008] Preferably, the conditions for cold plasma treatment are: The power is 75W, and the processing time is 1min~5min.

[0009] Preferably, the cold plasma treatment time is 3 minutes.

[0010] Preferably, the inert gas is any one of argon, helium, neon, and krypton.

[0011] Preferably, the oligosaccharide is galactooligosaccharide.

[0012] Preferably, the conditions for the glycosylation reaction are as follows: The reaction is carried out at 58℃~62℃ and relative humidity of 76%~80% for 2.5h~3.5h.

[0013] Preferably, the conditions for the glycosylation reaction are as follows: The reaction was carried out at 60℃ and 79% relative humidity for 3 hours.

[0014] Preferably, the buffer solution is PBS.

[0015] Preferably, the static incubation conditions are: 4℃, 16h~18h.

[0016] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a method for improving the functional properties of casein, comprising the following steps: S1: Dissolve casein in buffer solution to obtain a casein solution; S2: Treat the casein solution with cold plasma using an inert gas as the working gas, stir evenly after completion, and incubate to obtain a pretreated casein solution; S3: Add oligosaccharides to the pretreated casein solution, mix well, let stand, and freeze-dry to obtain a lyophilized powder of the mixture; S4: Perform glycosylation reaction on the lyophilized powder of the mixture to improve the functional properties of casein. Based on plasma pretreatment combined with glycosylation technology, this invention achieves functional modification of casein. This invention first opens the casein molecular structure and exposes amino sites through cold plasma, and then achieves efficient glycosylation under mild conditions. Compared with the prior art, the method described in this invention not only effectively reduces its IgE binding capacity and reduces the risk of allergies, but also significantly improves the solubility, emulsification, foaming properties, and thermal stability of casein.

[0017] Specifically: Emulsifying properties are significantly improved. Cold plasma pretreatment promotes protein unfolding, and combined with glycosylation, it significantly enhances the emulsifying activity and emulsion stability of casein. Foaming and foam-holding properties are enhanced. The introduction of hydrophilic groups into sugar molecules improves the protein's adsorption capacity at the gas-liquid interface, making the foam more stable. Solubility and water-holding capacity are improved: Glycosylation modification increases the hydrophilicity of the protein, reduces its aggregation tendency, and improves its application performance in complex food systems. Thermal stability is enhanced: The denaturation temperature of modified casein is increased, which is beneficial for maintaining function during high-temperature processing, such as pasteurization and UHT sterilization. Allergenicity is reduced: Glycosylation masks some IgE epitopes in casein, reducing the binding of allergens to immune cells and lowering the risk of allergies.

[0018] Furthermore, the method of this invention is mild, and compared with traditional high-temperature glycosylation, this invention can achieve efficient modification at medium and low temperatures, avoiding excessive protein denaturation or loss of function; it has high grafting efficiency, and cold plasma induces more reaction sites to be exposed, which greatly improves the glycosylation efficiency; it is green and environmentally friendly, requiring no additional chemical reagents, avoiding harmful residues, and conforming to the development trend of clean label food; it has strong controllability, and by adjusting the plasma power, processing time and sugar ratio, the properties of the product can be flexibly controlled to meet the needs of different foods.

[0019] The protein modification method described in this invention can be used to develop hypoallergenic dairy products, suitable for infant formula, functional dairy beverages, hypoallergenic yogurt, etc.; functional food additives, which can be used as natural emulsifiers, foaming agents or stabilizers in beverages, baked goods, and sports nutrition foods; it has great industrialization potential, as this method can be introduced into existing dairy processing technology and has the feasibility and economics for widespread application.

[0020] The method of this invention also overcomes the problems of low efficiency, high energy consumption, and insufficient modification of traditional heat treatment and Maillard reaction. It has obvious novelty and inventiveness, and the process is controllable, energy consumption is low, and it is suitable for industrial promotion. It has good practicality and application prospects. Attached Figure Description

[0021] Figure 1 The effect of different treatment conditions on casein solubility.

[0022] Figure 2 The effect of different treatment conditions on the emulsifying properties of casein. A: Emulsifying activity (EAI); B: Emulsifying stability (ESI).

[0023] Figure 3 The effect of different treatment conditions on the foaming properties of casein. A~C represent, in order: foam capacity, foam stability, and foam microstructure.

[0024] Figure 4 To investigate the effects of different treatment conditions on the binding capacity of casein IgE.

[0025] Figure 5 The particle size results for different products are shown. a: Particle size distribution; b: Average particle size.

[0026] Figure 6 The results show the ζ-potentials of different products.

[0027] Figure 7 The results show the surface hydrophobicity of different products.

[0028] Figure 8 This is the OES spectrum.

[0029] Figure 9 The content of free thiol groups in different products.

[0030] Figure 10 Secondary results analysis for different products. a: FTIR spectrum; b: relative content of secondary structures.

[0031] Figure 11 Tertiary results analysis for different products. a: Intrinsic fluorescence spectrum; b: Ultraviolet spectrum.

[0032] Figure 12 The effect of different plasma pretreatment times on casein grafting degree and browning intensity.

[0033] Figure 13 SDS-PAGE results for different products. Detailed Implementation

[0034] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0035] The inventive concept of this invention is as follows: Cold plasma is a non-thermal processing technology with advantages such as low energy consumption, gentle processing, and environmental friendliness. Particles such as reactive oxygen species and reactive nitrogen radicals generated by cold plasma can disrupt intermolecular forces within protein molecules, inducing protein structure unfolding, exposing more active sites, and providing conditions for subsequent modification reactions. Although cold plasma has been used in some protein modification applications, systematic research on the improvement of casein sensitization and function is still lacking, especially the combined effect of cold plasma and Maillard reaction in casein modification. Therefore, there is an urgent need for a green and efficient new technology to achieve functional enhancement and desensitization of casein.

[0036] Compared with the prior art, the improvements of the present invention are mainly reflected in the following aspects: 1. Different protein dissolution conditions: This invention uses biological buffer (PBS) to dissolve proteins. The buffer salt ions / stable pH help maintain the controllable unfolding of protein conformation, reduce irreversible aggregation, and provide a more stable chemical environment for subsequent exposure of glycosylation sites and improvement of interface functions (emulsification / foaming).

[0037] 2. Differences in plasma discharge medium and subsequent processing pathway: This invention uses inert argon gas as the working gas for DBD treatment and performs low-temperature overnight incubation after CP. Inert gas discharge primarily produces mild ROS with minimal RNS, and the overnight incubation facilitates protein conformation unfolding and site exposure, reducing the risk of excessive oxidation and flavor degradation. Existing technologies use air as the gas source and do not include overnight incubation. Air discharge simultaneously generates ROS and RNS, resulting in a more complex chemical modification spectrum of side chains and a higher risk of excessive oxidation.

[0038] 3. Different saccharification mode and process sequence: This invention first hydrates and mixes the powder, then freeze-dries it before performing constant temperature and humidity dry heat saccharification. Dry-state humidity control inhibits late Maillard and browning reactions, resulting in relatively higher protein grafting, lighter color, and fewer flavor byproducts. Furthermore, the mixed powder before saccharification facilitates storage and improves batch-to-batch reproducibility. Existing patents use a wet hot water bath, which is faster but more prone to advancing to deeper reactions, increasing the risk of color deepening and irreversible aggregation.

[0039] 4. This invention emphasizes multi-dimensional functional improvements such as emulsification, foaming, solubility, and thermal stability, and achieves a decreasing trend in IgE binding. This results in not only low allergenicity but also improved functionality, facilitating applications in specific products such as beverages, stabilizers, and foaming systems.

[0040] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0041] The list of abbreviations for this invention is shown in Table 1.

[0042] Table 1. List of abbreviations for this invention The reagents and instruments used in this invention are sourced from the following sources: Casein: Purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number S12002.

[0043] Dielectric barrier discharge plasma equipment: consists of a DBD-100 reactor and a CTP-2000K power supply.

[0044] Galacto-oligosaccharides: purchased from Maclean's, model G909325, purity 98%.

[0045] Example 1 A method for improving the functional properties of casein is as follows: S1: Dissolve casein in buffer solution to obtain casein solution.

[0046] Weigh out the casein product - casein, and disperse it in 20mM, pH 7.4 phosphate-buffered saline (PBS) to obtain a 20mg / mL casein solution.

[0047] S2: Using an inert gas as the working gas, the casein solution is subjected to cold plasma treatment. After the treatment is completed, the solution is stirred evenly and allowed to stand for incubation to obtain a pretreated casein solution.

[0048] The casein solution obtained in S1 was placed in the reactor of a dielectric barrier discharge plasma device for reaction, using argon as the working gas, maintaining a flow rate of 1 L / min, setting the output power to 75 W, and continuing for 3 min. After treatment, the mixture was stirred at 300 rpm for 2 h using a magnetic stirrer, and then allowed to stand overnight at 4 °C to ensure complete hydration, thus obtaining a pretreated casein solution.

[0049] During the cold plasma treatment in this step, active particles are generated through electron or heavy ion collisions to alter the protein structure of casein.

[0050] S3: Add oligosaccharides to the pretreated casein solution, mix well, let stand, and freeze dry to obtain a lyophilized powder of the mixture.

[0051] Galacto-oligosaccharides were added to the pretreated casein solution obtained in S2, stirred until homogeneous, and allowed to stand for 12 hours to ensure a sufficient proportion of galacto-oligosaccharides in the system. The dry matter mass ratio of galacto-oligosaccharides to casein was 1:2. Subsequently, the mixture was freeze-dried at -50℃ to obtain a lyophilized powder.

[0052] To achieve an efficient and controllable glycosylation reaction between galactooligosaccharides and casein, it is essential to ensure sufficient contact and uniform dispersion of both in solution. Therefore, this step involves mixing the two first. This step provides sufficient reducing end groups for the subsequent Maillard glycosylation reaction and, by controlling the amount of galactooligosaccharides added, avoids excessive sugar that could lead to severe browning and undesirable flavors, thus achieving a balance between the degree of glycosylation and the sensory quality of the product. Freeze-drying maximizes the preservation of the natural conformation of casein and the structural integrity of galactooligosaccharides, preventing protein denaturation or sugar degradation caused by overheating. Simultaneously, it yields a porous mixture powder with good dispersibility and a large specific surface area, which is beneficial for subsequent uniform and mild glycosylation reactions under controlled temperature and humidity conditions to obtain the final powder mixture.

[0053] S4: The lyophilized powder mixture was subjected to glycosylation reaction at 60°C and 79% relative humidity for 3 hours.

[0054] The lyophilized powder of the mixture obtained in step S3 was placed in a constant temperature and humidity drying oven at 60°C and 79% relative humidity for 3 hours. The glycosylated product was finally obtained and designated as CP+MR-3.

[0055] Based on a comprehensive consideration of Maillard reaction kinetics and water activity regulation, under suitable temperature and relative humidity conditions, a mild and controllable glycosylation reaction can occur between galactooligosaccharides and casein, thereby selectively improving the physicochemical and functional properties of casein without significantly reducing its nutritional value. On the one hand, a moderate temperature of 60°C provides sufficient energy to drive the initial stage of the Maillard reaction between reducing sugars and free amino groups in casein, while avoiding excessive browning and the excessive formation of advanced glycation end products (AGEs) under high-temperature conditions, thus balancing safety and nutritional value. On the other hand, by controlling the relative humidity at 79% to regulate the water activity of the system, the limited moisture in the powder can serve as a reaction medium to promote the migration and contact of reactant molecules, without causing excessive moisture that would inhibit the Maillard reaction or lead to powder agglomeration and reduced flowability.

[0056] Example 2 A method for improving the functional properties of casein is as follows: The cold plasma pretreatment time in S2 is 1 minute, and the rest of the process is exactly the same as in Example 1. The product is denoted as CP+MR-1.

[0057] Example 3 A method for improving the functional properties of casein is as follows: The cold plasma pretreatment time in S2 is 5 minutes, and the rest of the process is exactly the same as in Example 1. The product is denoted as CP+MR-5.

[0058] Comparative Example 1 A method for improving the functional properties of casein is as follows: S1: Dissolve casein in buffer solution to obtain casein solution.

[0059] The specific process is the same as S1 in Example 1.

[0060] S2: Using an inert gas as the working gas, the casein solution is subjected to cold plasma treatment. After the treatment is completed, the solution is stirred evenly and then allowed to stand for incubation.

[0061] The casein solution was placed in the reactor of the dielectric barrier discharge plasma device and reacted for 1 minute. The rest of the process was the same as S2 in Example 1. The product was designated as CP-1.

[0062] Comparative Example 2 A method for improving the functional properties of casein is as follows: S1: Dissolve casein in buffer solution to obtain casein solution.

[0063] The specific process is the same as S1 in Example 1.

[0064] S2: Using an inert gas as the working gas, the casein solution is subjected to cold plasma treatment. After the treatment is completed, the solution is stirred evenly and then allowed to stand for incubation.

[0065] The specific process is the same as S2 in Example 1, and the product is denoted as CP-3.

[0066] Comparative Example 3 A method for improving the functional properties of casein is as follows: S1: Dissolve casein in buffer solution to obtain casein solution.

[0067] The specific process is the same as S1 in Example 1.

[0068] S2: Using an inert gas as the working gas, the casein solution is subjected to cold plasma treatment. After completion, the solution is stirred evenly and then allowed to stand for incubation.

[0069] The casein solution was placed in the reactor of the dielectric barrier discharge plasma device for 5 minutes. The rest of the process was the same as S2 in Example 1. The product was designated as CP-5.

[0070] Comparative Example 4 A method for improving the functional properties of casein is as follows: Step S2 of Example 1 is missing, but everything else is exactly the same as in Example 1. The product is denoted as MR.

[0071] The products prepared in Examples 1-3 and Comparative Examples 1-4 were validated. In the following experiments, NC served as the control group, representing untreated natural casein. Details are as follows:

[0072] Experiment 1: Solubility test.

[0073] Compared with the control group, CP+MR-3 showed a 34.61% increase in solubility at pH 8.5, demonstrating better alkaline solubility. (See results below.) Figure 1 This result indicates that plasma pretreatment combined with glycosylation can effectively improve the solubility of casein and enhance its dispersion performance in different food systems.

[0074] like Figure 1 As shown, the solubility of natural casein is significantly higher under alkaline conditions compared to acidic conditions, mainly due to the isoelectric point of casein being approximately 4.6. Furthermore, compared to the NC control group, the CP-1, CP-3, and CP-5 groups treated with cold plasma generally showed a decreasing solubility trend within the pH range of 4.5–10.5. For the MR group treated solely with dry heat, its solubility increased within the alkaline pH range but decreased slightly under acidic conditions. Although glycosylation can induce some structural modification of CN, in an acidic environment, optimal molecular rearrangement is limited, and aggregation is more likely, thus only resulting in a slight increase in solubility.

[0075] Compared to NC, MR, CP-1, CP-3, and CP-5, the products of Examples 1-3 showed significantly improved solubility in the pH range of 2.5-10. Specifically, at pH 8.5, the solubility of CP+MR-3 increased by 34.61% compared to the NC group, indicating that the combined treatment of cold plasma and dry heat glycosylation can effectively enhance the solubility of CN. This increase in solubility is mainly attributed to the introduction of the hydrophilic oligosaccharide galactooligosaccharide. On the one hand, the grafting of galactooligosaccharides increases the overall polarity of the glycoprotein system, thereby inhibiting protein aggregation; on the other hand, cold plasma treatment can activate and partially unfold the protein structure, promoting the penetration of water and sugar molecules into the protein interior, further improving the solubility of the system. In addition, the enhancement of solubility is more significant under alkaline conditions, possibly because in an acidic environment, CN molecules are partially protonated and form a relatively compact conformation, thereby hindering the penetration and binding of water.

[0076] Experiment 2: Emulsification performance test.

[0077] Compared with NC, CP+MR-3 showed a 176.55% increase in emulsifying activity index and a 94.07% increase in emulsifying stability index. (See results below.) Figure 2 These results show that the synergistic effect of cold plasma pretreatment and glycosylation significantly enhances the emulsifying properties of casein, and the treated product is more suitable for use in dairy beverages and emulsified foods.

[0078] Figure 2 In the CP-5 group, compared to the NC group, an increase of 26.39% in EAI and 25.97% in ESI were observed. The MR obtained by conventional heating achieved 218.27 m compared to the NC group. 2 / g EAI; cold plasma-assisted MR further significantly improved this capacity, namely: the EAI and ESI of the CP+MR-3 group increased by 176.55% and 94.07%, respectively. This is likely due to the synergistic effect of cold plasma treatment and the addition of galactooligosaccharides, which together altered the conformation and surface charge of the protein. Protein unfolding promotes the formation of covalent bonds between sugars and proteins, while the increased sugar content and electrostatic charge further enhance the emulsifying properties. Nevertheless, the EAI and ESI values ​​of CP+MR-5 showed a decrease, but they were still significantly higher than those observed in the untreated and CP-treated samples alone. This may stem from prolonged cold plasma exposure, which may lead to oxidative degradation of key glycosylation groups and reduce glycosylation efficiency.

[0079] Experiment 3: Foaming performance test.

[0080] In this experiment, the foaming ability was characterized by foam capacity FC and foam stability FS; the microstructure of the foam was observed by allowing it to stand for different periods of time.

[0081] Compared to NC, CP+MR-3 achieved a foam capacity of 157.78% and improved foam stability by 48.82%. Microscopic observation revealed that CP+MR-1 produced finer and more uniformly distributed foam bubbles. This demonstrates that the method provided by this invention can effectively improve the foaming and foam-holding properties of casein.

[0082] Figure 3In the study, the FC (foaming capacity) of CP-3 was significantly improved, increasing from 145.55% of NC to 157.78%. This is because CP treatment accelerates the protein dissociation process, promoting the formation of a more flexible structure. This structural change accelerates protein adsorption at the air-water interface, thereby enhancing the foaming properties of casein. In contrast, the FS (foaming surface area) of CP-3 decreased after cold plasma treatment. The ordered and compact protein structure helps to form a highly elastic surface layer, which creates an effective barrier for FS. Therefore, the reduction in FS may be related to the disruption of chemical bonds between casein molecules, including hydrogen bonds and disulfide bonds. Plasma exposure can disrupt the protein network, weaken the foam wall, and reduce its resistance to external instabilities. Notably, the products of Examples 1-3 exhibited excellent foaming properties, especially FS. The FS of CP+MR-3 increased by 48.82%, potentially due to the enhanced grafting efficiency between GOS and CN promoted by plasma pretreatment. This synergistic enhancement comes from plasma-induced partial unfolding of the protein and exposure of active amino groups, which increases the availability of glycosylation sites. Subsequently, the Maillard reaction introduced hydrophilic carbohydrate chains, improving molecular flexibility and interfacial viscosity. In summary, these structural modifications contribute to the formation of a more cohesive and elastic interfacial film, thereby enhancing foam stability. Furthermore, the presence of sugars may contribute to enhanced foam performance by increasing the viscosity and solubility of the conjugates. Changes in foam microstructure show that, compared to other groups, the products of Examples 1-3 produced significantly more uniform foams with smaller sizes, exhibiting a slower rate of structural change, which corresponds to the results of FS and FC. In conclusion, the synergy of plasma pretreatment and MR is an effective method for improving protein foaming performance.

[0083] Experiment 4: Thermal stability test.

[0084] This experiment used differential scanning calorimetry (DSC) to detect the thermal denaturation characteristics. Compared with the control group, the denaturation temperatures of the products in Examples 1 to 3 increased by 15.95℃ to 18.51℃, indicating that the method of this invention can significantly enhance the thermal stability of casein, enabling it to maintain good structure and function under high-temperature processing conditions. The results are shown in Table 2.

[0085] Table 2 Effects of different treatment conditions on the thermal stability of casein Experiment 5: Allergy test.

[0086] The binding capacity of IgE to different products was detected using indirect ELISA. With increasing cold plasma pretreatment time, the IgE binding rate of the products from Examples 1 to 3 gradually decreased, with the IgE binding rates of CP+MR-3 and CP+MR-5 decreasing by 21.75% and 29.58%, respectively. This indicates that this method can effectively reduce the allergenicity of casein and enhance its application value in hypoallergenic dairy products.

[0087] Figure 4 In the study, with increasing cold plasma pretreatment time, the binding capacity of the products from Comparative Examples 1 to 3 to IgE significantly decreased, with CP-5 decreasing by 10.05%. This decrease in IgE binding capacity can be explained by alterations in casein antigenic epitopes. Reactive particles generated by cold plasma may interact with specific amino acid residues, impairing IgE recognition of linear epitopes and alleviating allergic reactions. Notably, after cold plasma pretreatment and glycosylation, the IgE binding capacity further decreased by 14.99% (CP+MR-1), 21.75% (CP+MR-3), and 29.58% (CP+MR-5), respectively, which may be attributed to the masking effect of reducing sugars. Furthermore, it was found that the IgE binding capacity of the products generated by cold plasma-assisted glycosylation was lower than that of the NC and MR groups.

[0088] The products prepared in Examples 1-3 and Comparative Examples 1-4 were characterized, and the results are as follows: 1. Particle size.

[0089] See results Figure 5 Compared to NC, the particle size of the products in Examples 1-3 was significantly reduced, from 309.2 nm to 248.25 ± 3.35 nm, and the particle size distribution became more uniform, exhibiting a single-peak distribution. This indicates that the combined effect of cold plasma pretreatment and saccharification effectively promoted the depolymerization and structural changes of casein. Plasma pretreatment, through the action of high-energy reactive species, disrupted the covalent bonds within the protein, enhanced the charge repulsion between protein molecules, and inhibited aggregation; while saccharification, through the introduction of glycans, further promoted protein depolymerization, reducing its particle size and making it more uniform. This treatment not only reduced the presence of larger particles but also improved the dispersibility and surface activity of the protein, providing strong support for subsequent functional improvements such as emulsification and foam stability. This phenomenon highlights the significant advantages of combined treatment in controlling protein particle size and improving protein functionality, especially in food processing and functional materials applications, where it has broad application potential. Stable pH and ionic strength (PBS system) to some extent prevented irreversible aggregation and promoted the controllable unfolding of the protein.

[0090] 2. Zeta potential.

[0091] See results Figure 6 The significant changes in the absolute zeta potential of the products in Examples 1 and 3 indicate that the introduction of glycans and plasma pretreatment together lead to an increase in the negative charge on the protein surface. This phenomenon suggests that, after plasma pretreatment is combined with the glycosylation reaction, the spatial shielding effect of glycans becomes the dominant factor, enhancing the coverage of the protein by the glycans and reducing the interactions between proteins. Furthermore, the increase in negatively charged groups and charge density during the glycosylation reaction further promotes the reaction, improves the surface charge of the protein, and thus enhances its functionality and stability.

[0092] 3. Surface hydrophobicity.

[0093] See results Figure 7 The H0 value is often used as an indicator of protein surface hydrophobicity. A higher H0 value usually means that more hydrophobic groups are exposed on the protein surface.

[0094] The H0 values ​​of the products in Examples 1-3 were significantly reduced. This change indicates that the combined effect of plasma treatment and saccharification resulted in exposed hydrophobic regions becoming sites for saccharification. Plasma pretreatment, through the action of high-energy electrons and free radicals, partially unfolded the CN structure, exposing hidden hydrophobic residues and thus increasing the H0 value. After saccharification, the introduction of glycans masked the surface hydrophobic groups through spatial shielding and the increase of hydrophilic groups, leading to a decrease in the H0 value. This phenomenon demonstrates that saccharification effectively promotes the covalent bonding of glycosides to proteins, alters the surface properties of proteins, further optimizes the efficiency of the saccharification reaction, and improves the functionality of the conjugate, while avoiding excessive structural changes.

[0095] This invention uses inert argon as the working gas for DBD treatment and performs overnight incubation at low temperature after CP. The inert gas discharge produces predominantly mild ROS and low levels of RNS. Combined with overnight incubation, this facilitates protein conformation unfolding and site exposure, reducing the risk of excessive oxidation and flavor degradation. Existing technologies use air as the gas source and do not include overnight incubation. Air discharge simultaneously generates ROS and RNS, resulting in a more complex chemical modification spectrum of side chains and a higher risk of excessive oxidation.

[0096] 4. OES emission spectrum.

[0097] OES analysis revealed the generation of various reactive oxygen and nitrogen species (RONS) during cold plasma treatment, such as hydroxyl radicals (·OH), nitrogen oxides (NOx), and singlet oxygen. These species are generated after the interaction of argon with water molecules and diffuse into the solution. A peak at 308 nm indicates the presence of ·OH, which is the main oxidizing medium in plasma treatment and promotes protein conformational unfolding and site exposure. The RONS generated by cold plasma exhibit different reactivity and lifetimes, playing a crucial role in subsequent saccharification reactions, enhancing protein grafting efficiency, while avoiding over-oxidation and reducing the formation of flavor byproducts. Results are shown below. Figure 8 The results showed that cold plasma pretreatment can enhance protein functionality through mild oxidation while ensuring the effectiveness of the saccharification reaction.

[0098] 5. Free thiol group.

[0099] In this invention, the treatments in Examples 1-3 significantly altered the content of free thiol groups (-SH) in the protein. With increasing plasma pretreatment time, the -SH content increased from 20.79 μmol / g to 23.87 μmol / g, indicating that the breaking of disulfide bonds promoted the exposure of -SH groups. This conformational change was due to oxidation induced by high-energy plasma particles, leading to structural relaxation of the protein. After saccharification, the free -SH content in the CP+MR-3 and CP+MR-5 groups decreased significantly, which may be related to the formation of disulfide bonds between -SH groups and other molecules, thus affecting the further progress of the saccharification reaction. Furthermore, the reaction of α-dicarbonyl compounds generated during saccharification with -SH groups also accelerated the reduction in -SH content. Overall, the combined treatment significantly promoted the exposure of -SH groups and controlled the change in -SH content through the progression of the saccharification reaction, thereby improving saccharification efficiency and grafting effect. The above results are shown in […]. Figure 9 .

[0100] 6. Secondary structure.

[0101] FT-IR analysis showed that the treatments in Examples 1-3 significantly altered the structure of casein and promoted the saccharification reaction. The products of Examples 1-3 exhibited a red shift and intensity decay in the amide I and amide II bands, indicating a rearrangement of the protein's secondary structure, particularly changes in the α-helix and β-sheet structures. This suggests that plasma treatment promoted protein conformation unfolding, exposing more amino groups and reaction sites, thereby enhancing the efficiency of the saccharification reaction. Furthermore, the α-helix content of the saccharified protein increased, indicating that plasma treatment increased protein flexibility and facilitated the saccharification reaction. Glycosyl grafting altered the ordered structure of the protein, promoting the transition to a disordered conformation and improving the efficiency of the saccharification reaction. These results are shown in [see attached figures]. Figure 10 .

[0102] 7. Three-level structure.

[0103] The treatments in Examples 1-3 significantly altered the tertiary structure of casein. Fluorescence and UV absorption spectroscopy analyses revealed that plasma pretreatment exposed aromatic amino acids in CN and induced a conformational change in tryptophan residues, making them more exposed to the reaction and thus enhancing the efficiency of the glycosylation reaction. The decrease in fluorescence intensity and redshift indicated that plasma treatment altered the protein structure and amino acid environment, increasing the availability of aromatic amino acids on the surface. After glycosylation, the protein structure further changed, resulting in stronger UV absorption peaks, especially in the plasma-glycosylation combined treatment group. The enhanced absorption peaks indicated that the glycosylation process promoted the binding of glycogroups to the protein, forming higher molecular weight glycoprotein complexes. These results are shown in […]. Figure 11 .

[0104] This invention involves first hydrating and mixing the powder, then freeze-drying it before subjecting it to constant temperature and humidity dry-heat saccharification. This dry-state, humidity-controlled process inhibits late-stage Maillard and browning reactions, resulting in higher protein grafting rates, lighter color, fewer flavor byproducts, and improved storage of the pre-saccharified powder, along with stronger batch-to-batch reproducibility. Existing patents utilize a wet hot water bath, which is faster but more prone to advancing into deeper reactions, increasing the risk of color deepening and irreversible aggregation.

[0105] 8. Grafting degree and browning intensity.

[0106] Grafting degree is an important indicator for evaluating the efficiency of saccharification reactions and is closely related to the reduction of available free amino acids. Using natural casein as material, it was subjected to cold plasma treatment for different times and Maillard reaction, with results as follows: Figure 12 As shown.

[0107] The grafting degree peaked at 25.8% after CP pretreatment at 3 min, which was 13.7% higher than that of the sample treated with Maillard reaction only (0 min treatment). This phenomenon may be attributed to the effect of active particles (such as ·OH or HO2) generated in the plasma, which promote the unfolding of casein, exposing more amino acids and thus promoting the saccharification reaction. This phenomenon often occurs in plasma-treated proteins, and the long-lived active species generated during plasma treatment do not affect peptide bond cleavage. In addition, the increased flexibility of the protein makes it easier for sugar molecules to access the reaction site, thereby enhancing the saccharification process. However, when the CP pretreatment time was extended to 5 min, the grafting degree decreased slightly, possibly due to excessive oxidation and protein aggregation. Excessive pretreatment may alter or degrade the protein structure, thereby inhibiting the improvement of saccharification efficiency. Furthermore, the browning intensity (DB) mainly reflects the color change of the Maillard reaction. The 420 nm absorbance of the conjugate generated by the combined treatment was significantly higher than that of the sample generated by conventional dry heat treatment. However, after 4 minutes of plasma pretreatment, the rate of increase in browning intensity slowed down, indicating that excessively long plasma pretreatment may have an adverse effect on the Maillard reaction. This may be due to the depletion of reactive amino groups on the protein surface and the occurrence of side reactions as the reaction progresses. The structure of the glycans also reduces the likelihood of sugar molecules approaching the reaction site, thereby slowing down the glycation process.

[0108] 9. SFDS-PAGE.

[0109] SDS-PAGE analysis showed that the products of Examples 1-3 had higher molecular weight glycoprotein complexes. After glycosylation, the protein bands shifted slightly upwards and became more diffuse, indicating that amino covalent modification occurred, demonstrating the covalent binding between GOS and the protein. Plasma treatment did not significantly affect the peptide chain integrity of CN, nor did it cause significant cross-linking or degradation, ensuring the stability of the protein backbone structure. During glycosylation, due to the low degree of glycosylation or insignificant molecular weight change, no significant band pattern differences were observed in SDS-PAGE. These results indicate that the combined treatment helps promote the glycosylation reaction and effectively enhances the protein's functionality, forming a stable glycoprotein complex. The above results are shown in [reference needed]. Figure 13 .

[0110] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0111] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for improving the functional properties of casein, characterized in that, The steps are as follows: S1: Dissolve casein in a buffer solution at pH 7.4 to obtain a casein solution; S2: Using an inert gas as the working gas, the casein solution is subjected to cold plasma treatment. After the treatment is completed, the solution is stirred evenly and allowed to stand for incubation to obtain a pretreated casein solution. S3: Add oligosaccharides to the pretreated casein solution, mix well, let stand, freeze dry, and obtain the freeze-dried powder of the mixture; the dry mass ratio of oligosaccharides to casein is 1:1~2; S4: Glycosylation of the lyophilized mixture powder can improve the allergenicity, solubility, emulsification, foaming properties and thermal stability of casein.

2. The method as described in claim 1, characterized in that, The conditions for cold plasma treatment are: The power is 75W, and the processing time is 1min~5min.

3. The method as described in claim 2, characterized in that, The cold plasma treatment time is 3 minutes.

4. The method as described in claim 1, characterized in that, The inert gas is any one of argon, helium, neon, and krypton.

5. The method as described in claim 1, characterized in that, The oligosaccharide is galactooligosaccharide.

6. The method as described in claim 1, characterized in that, The conditions for the glycosylation reaction are as follows: The reaction is carried out at 58℃~62℃ and relative humidity of 76%~80% for 2.5h~3.5h.

7. The method as described in claim 6, characterized in that, The conditions for the glycosylation reaction are as follows: The reaction was carried out at 60℃ and 79% relative humidity for 3 hours.

8. The method as described in claim 1, characterized in that, The buffer solution is PBS.

9. The method as described in claim 1, characterized in that, The conditions for static incubation are: 4℃, 16h~18h.

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