Dual-modification solubilizing process of walnut protein and high-protein nutritional paste prepared from dual-modification solubilizing process

Abstract

The invention discloses a dual-modification solubilizing process of plant-based walnut protein and high-protein nutritional paste prepared from the plant-based walnut protein, and belongs to the technical field of plant protein modification. The compound is prepared by carrying out high-pressure homogenization pretreatment on walnut protein extracted and separated from walnut meal and then carrying out glycosylation reaction on the pretreated walnut protein and pectin, wherein the mass ratio of the walnut protein to the pectin is 1: (0.25-0.35). Physical and chemical means are adopted for synergistic modification, and interaction among protein molecules is destroyed through high-pressure homogenization pretreatment, so that the structure of the walnut protein isolate is looser, and more sites are provided for glycosylation reaction; the glycosylation is that saccharide molecules and protein molecules are combined through covalent bonds to form a protein-polysaccharide compound, so that the solubility and the stability of the walnut protein isolate are greatly improved. The nutritional paste is prepared by taking the compound as a core, compounding erythritol according to the ratio of (4-10): 1 and adding a proper amount of calcium gluconate and vitamin D3, and the product is easy to dissolve and stable and conforms to the dietary guideline.

Description

Technical Field

[0001] The invention belongs to the technical field of plant protein modification, and in particular relates to a double-modified solubilization process of walnut protein and a high-protein nutritional paste thereof. Background Art

[0002] Walnut meal, as the main byproduct of walnut oil extraction, is rich in high-quality plant protein. These proteins not only have a balanced amino acid composition and are easy for human digestion and absorption, but are also hypoallergenic, making them very suitable as a high-quality protein source for people with lactose intolerance. Walnut protein isolate extracted from walnut meal has extremely high nutritional value, but due to the high temperature treatment during the oil extraction process and subsequent processing steps, the solubility of walnut protein isolate is greatly reduced.

[0003] In order to improve the solubility of walnut protein isolate extracted from walnut meal and make full use of this high-quality protein resource, researchers treat walnut protein isolate by chemical modification or enzymatic hydrolysis, in order to obtain walnut protein isolate with significantly improved solubility. CN119157196A discloses a method for improving the solubility of walnut protein isolate by coordinating a metal polyphenol network, including the preparation of a metal polyphenol network conjugate and the formation of a ternary graft of the conjugate and walnut protein isolate; the metal polyphenol network conjugate is a coordination chelation of caffeic acid and zinc ions; the formation of the ternary graft includes the formation of a hydrophilic ternary graft of the caffeic acid-zinc ion conjugate with different proportions of walnut protein isolate under pH cycle induction, thereby obtaining a walnut protein isolate with significantly improved solubility and rich in zinc. CN114317660A discloses a method for improving the utilization rate of walnut meal protein, which uses walnut meal as the main raw material, uses papain to hydrolyze walnut protein to a limited extent, and then uses an ultrafiltration system to obtain hydrolyzed products according to molecular weight to obtain peptides of different molecular weights. Finally, it is found that MW>30Kha is glycosylated with sugars of different molecular weights, and the prepared walnut peptides have better solubility, emulsification properties, and antioxidant properties than other molecular weight polypeptides. CN112795611A discloses a method for preparing walnut protein polypeptides from insoluble proteins, which promotes the conversion of insoluble walnut protein into soluble walnut protein polypeptides through grinding, specific proportions of pepsin and alkaline protease, and ultrasonic enzymolysis, thereby increasing the content of small molecules in the polypeptide and improving the utilization rate of walnuts.

[0004] By searching domestic and foreign literature, it was found that the existing methods for improving walnut protein isolate are relatively complicated and not conducive to large-scale industrial production. Summary of the invention

[0005] In view of the deficiencies in the prior art, the first object of the present invention is to provide a walnut protein isolate-pectin covalent complex with a simple preparation process, which not only solves the problem of poor solubility of walnut protein isolate in walnut meal, but also maintains its original nutritional value.

[0006] In order to achieve the above technical objectives, the present invention aims at the problem of poor solubility of walnut protein isolate in walnut meal, adopts physical (high-pressure homogenization modification) and chemical means (glycosylation reaction) for coordinated treatment, and successfully prepares a soluble walnut protein isolate-pectin covalent complex.

[0007] Specifically, the object of the present invention is achieved as follows: a walnut protein isolate-pectin covalent complex is prepared by saccharification reaction of walnut protein extracted and separated from walnut meal with pectin after high-pressure homogenization pretreatment, wherein the mass ratio of the walnut protein isolate to pectin is 1: (0.05-0.35).

[0008] It should be noted that high-pressure homogenization pretreatment can destroy the interaction between protein molecules and make its structure looser, which is not only conducive to improving solubility, but also provides more sites for glycosylation reactions; glycosylation is to form a protein-polysaccharide complex by covalently bonding carbohydrate molecules to protein molecules, thereby further improving solubility and stability.

[0009] Further preferably, in the walnut protein isolate-pectin covalent complex as described above, the mass ratio of walnut protein to pectin is 1:(0.25-0.35), that is, the added amount of pectin is 25%-35% w / w.

[0010] Further preferably, in the walnut protein isolate-pectin covalent complex as described above, the conditions for high-pressure homogenization pretreatment are: adding water to prepare a walnut protein dispersion with a concentration of 1% to 5% (w / v), the homogenization pressure is 200 to 1000 bar, and the homogenization time is 4 to 10 minutes. Still further preferably, when high-pressure homogenization is performed during the preparation process, a better pressure range and homogenization times can be selected to achieve the best effect, wherein the homogenization pressure is preferably 700 to 800 bar, the homogenization time is preferably 4 to 6 minutes, and it is completed in 2 times.

[0011] Further preferably, the walnut protein isolate-pectin covalent complex as described above, wherein the conditions for the glycosylation reaction are: adjusting the pH of the glycosylation reaction system to 3-8 (preferably pH=7.0), the reaction temperature to 50-80°C, and the reaction time to 60-480 min. Still further preferably, the walnut protein isolate-pectin covalent complex as described above, wherein the conditions for the glycosylation reaction are: adjusting the pH of the reaction system to 7.0, the reaction temperature to 60°C, and the reaction time to 60 min.

[0012] In a preferred embodiment of the present invention, for the walnut protein isolate-pectin covalent complex as described above, the reaction product is subjected to vacuum freeze-drying. The drying temperature is -65 to -72 °C, the vacuum degree is 5 to 10 Pa, the drying time is 24 to 48 h, and after drying, it is ultra-finely pulverized and sieved through a 120- to 140-mesh sieve.

[0013] In a preferred embodiment of the present invention, for the walnut protein isolate-pectin covalent complex as described above, the walnut protein therein uses the walnut meal after oil extraction as the raw material and adopts an extraction process of alkali extraction and acid precipitation to separate the walnut protein from the walnut meal. This extraction process includes steps such as crushing, leaching, and centrifugal separation to ensure that the purity of the extracted walnut protein isolate reaches more than 80%.

[0014] In addition, the present invention also provides a double-modification solubilization process for walnut protein, and this process includes the following steps:

[0015] (1) Take the walnut meal after oil extraction and obtain walnut protein isolate through the extraction process of alkali extraction and acid precipitation.

[0016] (2) Prepare the extracted walnut protein isolate into a walnut protein dispersion with a concentration of 1% - 5% (w / v) by adding water, and then perform high-pressure homogenization. The homogenization pressure is 200 - 1000 bar, the total homogenization time is 4 - 10 min, and it is homogenized 1 - 6 times in total to obtain a homogenized walnut protein isolate dispersion.

[0017] (3) After homogenization, add pectin accounting for 5% - 35% of the mass of the walnut protein isolate, stir evenly, adjust the pH of the reaction system to 3 - 8, and react at 50 - 80 °C for 60 - 480 min. After the reaction, cool it to room temperature to obtain a walnut protein isolate-pectin covalent complex solution.

[0018] (4) Perform vacuum freeze-drying treatment on the walnut protein isolate-pectin covalent complex solution. The drying temperature is -65 to -72 °C, the vacuum degree is 5 to 10 Pa, the drying time is 24 to 48 h, and after drying, it is ultra-finely pulverized and sieved through a 120- to 140-mesh sieve to obtain a walnut protein isolate-pectin covalent complex powder.

[0019] Based on the above walnut protein isolate-pectin covalent complex, the second object of the present invention is to provide an easily soluble plant-based high-protein walnut nutritional paste. This nutritional paste uses the modified walnut protein isolate-pectin covalent complex as the main raw material and is prepared through scientific formula design. It not only has excellent solubility and stability, but also is rich in high-quality plant protein and calcium elements, and can meet the diverse needs of consumers for nutritional supplements. At the same time, the development of the nutritional paste product also provides a new way for the effective utilization of walnut meal resources and environmental protection.

[0020] Therefore, the second object of the present invention is achieved as follows: a soluble plant-based walnut high-protein nutritional paste contains the above-mentioned walnut protein isolate-pectin covalent complex and erythritol, wherein the mass ratio of the walnut protein isolate-pectin covalent complex to erythritol is (4-10):1.

[0021] Further preferably, the above-mentioned soluble plant-based walnut high-protein nutritional paste also contains calcium gluconate and vitamin D 3 The nutritional paste can meet the diverse needs of different consumers for nutritional supplements by adding appropriate amounts of calcium and vitamins. When the paste is prepared with water for consumption, the material-liquid ratio is preferably 1:9-1:10 (w / v).

[0022] Compared with the prior art, the advantages and progress of the walnut protein isolate-pectin covalent complex and nutritional paste prepared by the present invention are mainly reflected in the following aspects:

[0023] (1) Raw material utilization: The traditional method does not fully utilize the walnut meal produced by walnut oil extraction. The present invention utilizes walnut meal to extract walnut protein isolate, turning waste into treasure, improving the utilization rate of walnut processing resources and reducing production costs.

[0024] (2) Protein modification: Traditional methods have poor effects on the poor solubility of walnut protein isolate. The present invention adopts physical and chemical means for synergistic modification. The interaction between protein molecules is destroyed by high-pressure homogenization pretreatment, making the structure of walnut protein isolate looser and providing more sites for glycosylation reaction. Glycosylation is to form a protein-polysaccharide complex by covalently bonding carbohydrate molecules to protein molecules, which greatly improves the solubility and stability of walnut protein isolate.

[0025] (3) Product performance: Calcium-containing nutritional products on the current market have the problems of poor taste and difficulty in digestion and absorption. The nutritional paste prepared by the present invention is not only rich in nutrition, but also has a delicate taste and is easy to digest and absorb by controlling parameters and the ratio of main and auxiliary materials. In addition, calcium and vitamins are added to meet diverse nutritional needs.

[0026] (4) Preparation process: The present invention ensures the quality and performance of the product through specific process steps, such as high-pressure homogenization, coordinated glycosylation modification, and vacuum freeze-drying combined with ultrafine grinding. Compared with traditional processes, it is more refined and retains the nutritional value of the product to the greatest extent.

[0027] In summary, compared with the prior art, the present invention shows significant beneficial effects in improving the solubility of walnut protein isolate, maintaining nutritional value, optimizing taste and system stability, achieving effective utilization of resources and broadening the application field, and provides a solution for the development and utilization of walnut meal resources and the health needs of consumers. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a relationship diagram between different homogenization pressures and the solubility and particle size of walnut protein isolate in Example 2;

[0029] Figure 2 This is a relationship diagram between different homogenization times and the solubility and particle size of walnut protein isolate in Example 3;

[0030] Figure 3 This is a relationship diagram between different polysaccharide types and the solubility and particle size of walnut protein isolate in Example 4;

[0031] Figure 4 This is a graph showing the relationship between the pectin addition ratio and the solubility and particle size of the walnut protein isolate in Example 5;

[0032] Figure 5 The relationship between different heating times and the solubility and particle size of walnut protein isolate in Example 6;

[0033] Figure 6 The relationship between different heating temperatures and the solubility and particle size of walnut protein isolate in Example 7;

[0034] Figure 7 The effect of different homogenization pressures on the Zeta potential of walnut protein isolate in Example 2;

[0035] Figure 8 The effect of different homogenization times on the Zeta potential of walnut protein isolate in Example 3;

[0036] Fig. 9 The effects of different polysaccharide types on the Zeta potential of walnut protein isolate in Example 4;

[0037] Fig.10 The effect of the pectin addition ratio on the Zeta potential of walnut protein isolate in Example 5;

[0038] Fig.11 The effect of different heating time on the Zeta potential of walnut protein isolate in Example 6;

[0039] Fig.12 The effect of different heating temperatures on the Zeta potential of walnut protein isolate in Example 7;

[0040] Fig.13 This is the SEM analysis of the walnut protein isolate-pectin covalent complex in Example 8;

[0041] Fig.14 Determination of the grafting degree of the walnut protein isolate-pectin covalent complex in Example 8;

[0042] Fig.15Comparison of centrifugal sedimentation rates of walnut protein nutrient pastes in different formulation groups in Example 9;

[0043] Fig.16 This is the Zeta potential analysis of the walnut protein nutritional paste with different formula groups in Example 9.

[0044] Among them: CK is the unmodified walnut protein isolate as the control group, HPH is the single high-pressure homogenization group, and HPH-GM is the high-pressure homogenization synergistic glycosylation modification group. DETAILED DESCRIPTION

[0045] The following examples are used to further describe the implementation process and beneficial effects of the method of the present invention. The test examples are only for illustrative purposes and do not limit the scope of protection of the present invention. At the same time, obvious changes made by ordinary technicians in this field based on the examples are also included in the scope of the present invention.

[0046] Example 1

[0047] This embodiment utilizes walnut meal resources produced during walnut oil extraction, improves the solubility of walnut protein isolate by modification technology, and uses walnut protein isolate-pectin covalent complex as the main raw material to prepare soluble walnut high-protein calcium-containing nutritional paste, and its production process includes the following steps:

[0048] S1. Take the walnut meal after oil pressing, extract walnut protein isolate by alkali extraction and acid precipitation method, and the purity reaches 85%.

[0049] S2. The extracted walnut protein isolate was fully mixed with water to obtain a 1% (w / v) walnut protein dispersion. The homogenization pressure was set to 700 bar, the homogenization time was 2.5 min each time, and the homogenization was performed twice to obtain a homogenized walnut protein isolate dispersion.

[0050] S3. After homogenization, add 25% (w / w) pectin of walnut protein isolate (walnut protein isolate: pectin = 1:0.25), adjust the pH of the reaction system to 7.0, stir evenly, react at 60°C for 60 minutes, and cool to room temperature after the reaction to obtain a walnut protein isolate-pectin covalently bound complex solution.

[0051] S4. The reaction product is subjected to vacuum freeze drying at a drying temperature of -70°C, a vacuum degree of 5 Pa, and a drying time of 24 hours. After drying, the product is crushed by an ultrafine grinder and passed through a 120-140 mesh sieve to obtain a walnut protein isolate-pectin covalent complex powder.

[0052] S5. According to the intake standard, each pack of walnut protein nutrition paste contains walnut protein isolate-pectin covalent compound powder, erythritol, calcium gluconate and vitamin D. 3The mass ratio is (2800mg~3000mg):(400mg~600mg):50mg:0.0003mg to make a soluble plant-based walnut high-protein nutritional paste, and the formula design conforms to the standards of "Dietary Guidelines for Chinese Residents (2016)".

[0053] Example 2

[0054] This experimental example provides a walnut protein nutritional paste production process, the raw materials and steps are basically the same as those in Example 1, the difference is that: the homogenization pressure in step S2 is modified to 400bar, 500bar, 600bar, 700bar, 800bar, and 900bar, respectively, and by testing indicators such as solubility, it is optimized that 700bar is the best homogenization pressure.

[0055] Example 3

[0056] This experimental example provides a walnut protein nutritional paste production process, and its raw materials and steps are basically the same as those in Example 1, except that: under the condition that the homogenization pressure in step S2 is 700 bar, the homogenization times are modified to 1, 2, 3, 4, 5, and 6 times respectively, and the optimal homogenization times after optimization is 2 times.

[0057] Example 4

[0058] This experimental example provides a walnut protein nutritional paste production process, and its raw materials and steps are basically the same as those in Example 1, except that: in step S3, polysaccharides such as pectin, xanthan gum, guar gum, flaxseed gum, carrageenan, and tamarind gum are used respectively, and the effects of different polysaccharides on the solubility of walnut protein are compared. The best polysaccharide after optimization is pectin.

[0059] Example 5

[0060] This experimental example provides a walnut protein nutritional paste production process, and its raw materials and steps are basically the same as those in Example 1, except that: the pectin addition amount in step S3 is modified to 5%, 10%, 15%, 20%, 25%, 30%, and 35% (w / w), respectively, and the optimized optimal pectin addition amount is 25%.

[0061] Example 6

[0062] This experimental example provides a walnut protein nutritional paste production process, and its raw materials and steps are basically the same as those in Example 1, except that: the heating time in step S3 is modified to 60, 120, 180, 240, 300, 360, 420, and 480 min, respectively, and the optimized optimal heating time is 60 min.

[0063] Example 7

[0064] This experimental example provides a walnut protein nutritional paste production process, and its raw materials and steps are basically the same as those in Example 1, except that: the heating temperature in step S3 is modified to 40, 50, 60, 70, and 80°C for reaction for 60 minutes, and the optimized optimal heating temperature is 60°C.

[0065] Example 8

[0066] This example is based on a single factor experiment and conducts an orthogonal experiment (formula grouping). The amount of modified compound added, erythritol added, and water added were selected as influencing factors, and three levels were set for each: the amount of modified compound added was 56%, 58%, and 60%; the amount of erythritol added was 8%, 10%, and 12%; the amount of water added was 90mL, 95mL, and 100mL. The specific formula groups are as follows:

[0067] Nutritional paste formula 1: modified complex addition amount 56%, erythritol addition amount 8%, water addition amount 90mL;

[0068] Nutritional paste formula 2: modified complex addition amount 56%, erythritol addition amount 10%, water addition amount 95mL;

[0069] Nutritional paste formula 3: modified complex addition amount 56%, erythritol addition amount 12%, water addition amount 100mL;

[0070] Nutritional paste formula 4: modified complex addition amount 58%, erythritol addition amount 8%, water addition amount 95mL;

[0071] Nutritional paste formula 5: modified complex addition amount 58%, erythritol addition amount 10%, water addition amount 100mL;

[0072] Nutritional paste formula 6: modified complex addition amount 58%, erythritol addition amount 12%, water addition amount 90mL;

[0073] Nutritional paste formula 7: modified complex addition amount 60%, erythritol addition amount 8%, water addition amount 100mL;

[0074] Nutritional paste formula 8: modified complex addition amount 60%, erythritol addition amount 10%, water addition amount 90mL;

[0075] Nutritional paste formula 9: modified complex addition amount 60%, erythritol addition amount 12%, water addition amount 95mL.

[0076] Preparation steps: According to the above 9 groups of nutritional paste formulas, weigh walnut protein isolate-pectin covalent complex powder, erythritol, calcium gluconate, and vitamin D3 respectively, add water at 100 °C with the corresponding volume, and mix thoroughly to obtain 9 groups of nutritional paste samples. Through comprehensive evaluation of the centrifugal precipitation rate and Zeta potential, determine the optimal formula to further verify the technical effect of the nutritional paste of the present invention in terms of stability.

[0077] Example 9: Experimental verification

[0078] In summary, through the process optimization of Examples 1-8 ( Figure 1-Figure 13 ), combined with the stability analysis of the walnut protein nutritional paste formula ( Figure 14-15 ):

[0079] (1) Solubility analysis:

[0080] The influence of different homogenization pressures. As can be seen from Figure 1 , the particle size of the control group (CK) is the smallest. During HPH treatment, the particle size first rises rapidly with the increase of pressure and then fluctuates to maintain a relatively high level; the particle size change trend of HPH-GM treatment is similar, but the value is slightly lower because HPH causes particle aggregation, while the pectin in HPH-GM can inhibit excessive aggregation. In terms of solubility, CK is the lowest, the solubility of HPH treatment first increases and then decreases, reaching a relatively high value at about 500 bar; the solubility of HPH-GM treatment is generally higher than that of HPH, and it is relatively high at 400 bar and fluctuates subsequently. Appropriate HPH increases the protein contact area and improves solubility, while too high pressure reduces solubility due to particle aggregation. The pectin in HPH-GM increases hydrophilicity and improves solubility.

[0081] The influence of different homogenization times. As can be seen from Figure 2 , the particle size of CK is the smallest. Under the HPH-700 bar treatment, the particle size first rises rapidly and then stabilizes with the increase of homogenization times; the particle size change trend of HPH(700 bar)-GM treatment is similar but the value is slightly lower. In terms of solubility, CK is the lowest, the solubility of HPH-700 bar treatment first increases and then fluctuates, and the solubility of HPH(700 bar)-GM treatment is generally higher and fluctuates. Appropriate homogenization increases the contact area and improves solubility, while too much reduces it due to particle aggregation. The pectin in HPH(700 bar)-GM increases hydrophilicity and improves solubility.

[0082] The influence of different polysaccharide types. As can be seen from Figure 3It can be seen that CK has the smallest particle size, which increases after HPH treatment. The particle size of HPH-GM treatment varies depending on the type of polysaccharide. The particle size of pectin treatment is the largest because HPH causes the particles to aggregate and pectin is more likely to promote particle interaction. In terms of solubility, CK is relatively low and increases after HPH treatment. In HPH-GM treatment, pectin and xanthan gum have higher solubility, while carrageenan has the lowest solubility because HPH destroys the protein structure. The hydrophilicity and molecular structure differences of different polysaccharides after modification affect the solubility.

[0083] The effect of different pectin ratios is determined by Figure 4 It can be seen that the particle size of CK and HPH-pH=7 treatments is smaller. In the HPH-GM treatment, when the pectin ratio increases from 5% to 35%, the particle size first increases and then stabilizes. The particle size of GM-25%-pectin treatment is higher in the early stage and stabilizes at 0.45-0.5μm in the later stage, because HPH causes the particles to aggregate and the pectin ratio affects the interaction between particles. In terms of solubility, CK is lower and HPH-pH=7 treatment improves it. In the HPH-GM treatment, the solubility increases as the pectin ratio increases, and the highest at 35%, because HPH increases the contact area and the increase in the pectin ratio enhances the hydrophilicity of the system.

[0084] The effect of different heating times is Figure 5 It can be seen that the particle size of the CK group is 0.35μm as the control. The particle size of the Heating-60℃ group is the smallest, indicating that simple heating reduces the particle size of walnut protein. The particle size of the HPH-700bar group increases, indicating that high-pressure homogenization causes protein aggregation. The particle size of the HPH-GM series group is larger than that of the CK and simple heating groups, and is regulated by process parameters such as temperature and time. In terms of solubility, the CK and Heating-60℃ groups are lower, and it is difficult to improve without treatment or simple heating. The HPH-700bar group has an improvement but the effect is limited. The solubility of most groups in the HPH-GM series has been significantly improved, but in groups such as HPH-GM-80℃-3h, the solubility has decreased due to excessive treatment that damages the protein-polysaccharide structure due to the continuous increase in time and temperature.

[0085] The influence of different heating temperatures is Figure 6 It can be seen that CK has the smallest particle size, and the particle size treated with HPH-700bar first increases and then fluctuates slightly with the increase of temperature. The particle size treated with HPH-GM is higher than CK and changes less and is more stable, because the temperature intensifies the aggregation of HPH particles, and the pectin in HPH-GM plays a dispersing and stabilizing role. In terms of solubility, CK is lower, and the solubility of HPH-700bar treatment increases with the increase of temperature. The solubility of HPH-GM treatment is initially higher than the former, but decreases with the increase of temperature, because HPH relies on temperature to destroy the structure and enhance dissolution, and the high temperature in HPH-GM causes pectin and other substances to react unfavorably to dissolution.

[0086] (2) Zeta potential analysis

[0087] The influence of different homogenization pressures is given by Figure 7 It can be seen that the absolute value of Zeta potential of CK is the smallest. The potential of single HPH treatment is maintained at -40mV from 400bar to 900bar, with no significant difference. The absolute value of potential of HPH-GM treatment increases significantly, fluctuates at -60mV, and there are differences under different pressures. Because HPH mainly changes the physical state of particles, it has little effect on the charge properties; the glycosylation of pectin in HPH-GM introduces charged groups to reduce the potential, and the homogenization pressure affects the binding of pectin to particles and the molecular conformation, thereby affecting the charge distribution.

[0088] The influence of different homogenization times is given by Figure 8 It can be seen that the potential of CK and HPH-700bar treatment is -40mV and stable without difference; the potential of HPH(700bar)-GM treatment is -60mV, and there is no difference between different times. Because HPH-700bar mainly changes the physical state, it has little effect on the charge; the pectin glycosylation in HPH(700bar)-GM introduces charged groups to reduce the potential, and the number of homogenization times has little effect on the charge distribution.

[0089] The effects of different polysaccharide types are Fig. 9 It can be seen that the potentials of CK, HPH-700bar, and Heating-60℃ treatments are similar, at -40mV; the potential of HPH-GM treatment varies depending on the type of polysaccharide, with the lowest potential of pectin treatment at -60mV, and the potentials of other polysaccharide treatments ranging from -20mV to -40mV. Because the first three treatments have little effect on the charge, pectin in HPH-GM has many charged groups, and the synergistic effect significantly reduces the potential, while other polysaccharides have fewer charged groups, and the degree of potential reduction varies.

[0090] The effect of different pectin ratios is determined by Fig.10 It can be seen that the potentials of CK, HPH-700bar and GM-pectin 25% treatments are similar, at -40mV; in the HPH-GM treatment, the pectin ratio increased from 5% to 35%, and the potential first decreased and then fluctuated, reaching the lowest at 20%. Because the first three have little effect on the charge, the low proportion of pectin in HPH-GM increases the charged groups and reduces the potential, and at a high proportion, the molecular interaction changes the charge distribution, resulting in potential fluctuations.

[0091] The effect of different heating times is Fig.11It can be seen that the Zeta potential value of CK is -40mV. The absolute values ​​of the Zeta potential of the CK group and the HPH-700bar group are small, indicating that the surface charge density of the walnut protein particles in these two groups is low and the stability of the system is relatively poor. The absolute value of the Zeta potential in the HPH-GM series group increased significantly, and the stability was improved. Therefore, only the high-pressure homogenization (HPH) treatment has little effect on the charge, while the high-pressure homogenization synergistic glycosylation (HPH-GM) treatment can introduce charged groups. The increase in temperature intensifies the glycosylation reaction, introduces more charged groups, increases the absolute value of the Zeta potential, enhances the electrostatic repulsion between particles, and improves stability.

[0092] The influence of different heating temperatures is Fig.12 It can be seen that the Zeta potential values ​​of CK and HPH-700bar treatments are similar, at -40mV; the potential of GM-25% treatment is slightly lower. Under HPH-GM treatment, the heating temperature increases from 30℃ to 80℃, and the Zeta potential value generally decreases first and then stabilizes. The absolute value of the Zeta potential value is the largest at 50℃. CK and HPH-700bar treatments have little effect on the surface charge of the particles, and the potentials are similar; GM-25% alone introduces certain charged groups, causing the potential to drop slightly; in HPH-GM treatment, heating at low temperatures promotes the structural unfolding of pectin glycosylation products, exposing more charged groups and reducing the Zeta potential; after the temperature rises to a certain level, the structural changes of pectin and other components tend to stabilize, the number and distribution of charged groups no longer change significantly, and the Zeta potential also tends to stabilize.

[0093] (3) Scanning electron microscopy analysis

[0094] Depend on Fig.13 It can be seen that the scanning electron microscope image shows that the unmodified walnut protein isolate particles are severely aggregated and the surface structure is tight; after being treated with the optimal process HPH-GM (homogenization 700bar, 25% pectin, heating at 60°C for 60min), the dispersion of the particles is significantly improved, and the surface presents a porous and loose structure, which intuitively proves that the process destroys the original aggregation structure of the protein and increases the hydrophilic sites, which is consistent with the solubility improvement result.

[0095] (4) Analysis of the grafting degree of covalent complexes

[0096] Depend on Fig.14 It can be seen that in the HPH-GM modification process, the grafting degree is significantly affected by the heating temperature and time. At 50-60℃, the grafting degree is higher when heated for 1h, while at 70-80℃, the grafting degree decreases when heated for 1h. At the same temperature, the grafting degree of heating for 3h is lower than that of heating for 1h, and the grafting degree is the lowest when heated at 80℃ for 3h.

[0097] (4) Analysis of centrifugal sedimentation rate and Zeta potential of walnut protein nutrient paste

[0098] The nutrient paste samples were prepared according to the ninth group of recipes in Example 8. The centrifugal sedimentation rate ( Fig.15 ) and Zeta potential ( Fig.16 ) Comprehensive evaluation. The results showed that the centrifugal sedimentation rate of the control group (CK) was 19.01%. After the formula design, the lowest sedimentation rate of the walnut protein nutrient paste reached 7.96% (a decrease of 58%). Combined with the Zeta potential (-54.4mV), formula 5 (58% complex + 10% erythritol + 95mL water) was determined to be the best, with a sedimentation rate of 6.16%, a protein content of 2.361g / 5g, and a calorie content of 10.15kcal / 5g, which meets the standards of GB5009.5-2016 and the Dietary Guidelines for Chinese Residents.

[0099] In summary, the soluble walnut protein isolate nutrient paste provided by the embodiment of the present invention has greatly improved solubility characteristics after synergistic modification. Comprehensive analysis determined that under the conditions of 2 homogenization times, 700 bar homogenization pressure, pectin as the polysaccharide type, 25% polysaccharide ratio, 60°C heating temperature, and 1 hour heating time, the solubility of the walnut protein isolate-pectin modified complex was 45.16%, an increase of 152% over the 17.89% of the control group. After formula design, the centrifugal sedimentation rate of the walnut protein nutrient paste dropped from 19.01% to 7.96%, a decrease of 58%. After 9 groups of formula verification ( Figure 14-15 ), the product has solubility, stability and nutritional balance, which improves the solubility and stability of the product.

[0100] Beneficial Effects

[0101] 1. Improved solubility: High-pressure homogenization synergistic glycosylation modification (HPH-GM) increased the solubility of walnut protein isolate to 45.16%, an increase of 152% over the control group (17.89%), solving the problem of poor solubility.

[0102] 2. Enhanced stability: The centrifugal sedimentation rate dropped from 19.01% to 7.96%, the absolute value of Zeta potential increased by 50% (-54.4mV), and the electrostatic repulsion between particles was enhanced, ensuring the stability of the nutrient paste during storage and use.

[0103] 3. Structure and reaction optimization: Scanning electron microscopy confirmed that the optimal process made the protein structure loose and porous; the grafting degree test showed that the protein and pectin were fully covalently bonded, providing dual support for improving solubility and stability from the structural and reaction levels.

[0104] 4. Process advantages: Different from the existing complex processes (such as the preparation of metal polyphenol network in CN119157196A), the process parameters of the present invention are clear (700 bar homogenization twice, 25% pectin, 60°C reaction for 60 min, solubility 45.16%, precipitation rate 7.96%), simple and easy to control, and suitable for industrial production (compared with the existing technology: CN119157196A requires metal coordination and the process is complicated).

[0105] 5. Nutritional optimization: Each serving of calcium-containing nutritional paste contains 2.8-3.0g of walnut protein isolate-pectin covalent complex, 4.5mg of calcium (calcium gluconate), and vitamin D 3 It is 0.3μg, which is in line with the dietary guidelines, taking into account solubility, stability and nutritional balance, and meets the nutritional standards of the "Dietary Guidelines for Chinese Residents".

Claims

1. A walnut protein isolate-pectin covalent complex, which is prepared by saccharification reaction of walnut protein extracted and separated from walnut meal with pectin after high-pressure homogenization pretreatment, wherein the mass ratio of the walnut protein to the pectin is 1:(0.05-0.35); the preferred mass ratio is 1:(0.25-0.35).

2. The walnut protein isolate-pectin covalent complex according to claim 1, characterized in that The conditions of the high-pressure homogenization pretreatment are: adding water to prepare a walnut protein dispersion with a concentration of 1% to 5% (w / v), a homogenization pressure of 200 to 1000 bar, and a homogenization time of 4 to 10 minutes.

3. The walnut protein isolate-pectin covalent complex according to claim 2, characterized in that The homogenization pressure is 700-800 bar, the homogenization time is 4-6 minutes, and it is completed in 2 times.

4. The walnut protein isolate-pectin covalent complex according to claim 1, characterized in that The conditions of the glycosylation reaction are: adjusting the pH of the reaction system to 3-8, the reaction temperature to 50-80° C., and the reaction time to 60-480 min.

5. The walnut protein isolate-pectin covalent complex according to claim 4, characterized in that: The conditions of the glycosylation reaction are: adjusting the pH of the reaction system to 7.0, the reaction temperature to 60° C., and the reaction time to 60 min.

6. The walnut protein isolate-pectin covalent complex according to any one of claims 1 to 5, characterized in that: The reaction product is vacuum freeze-dried, the drying temperature is -65 to -72°C, the vacuum degree is 5 to 10 Pa, the drying time is 24 to 48 hours, and after drying, it is ultrafinely ground and passed through a 120 to 140 mesh sieve.

7. The walnut protein isolate-pectin covalent complex according to any one of claims 1 to 5, characterized in that: The walnut protein is obtained by using walnut meal after oil pressing as raw material and extracting and separating it by using alkali extraction and acid precipitation method.

8. A double-modified solubilization process for walnut protein, the process comprising the following steps: (1) extracting walnut meal after oil pressing by alkali extraction and acid precipitation to obtain walnut protein isolate; (2) adding water to the extracted walnut protein isolate to prepare a walnut protein dispersion with a concentration of 1% to 5% (w / v), and then performing high-pressure homogenization at a homogenization pressure of 200 to 1000 bar for a total homogenization time of 4 to 10 minutes, and homogenizing 1 to 6 times in total to obtain a homogenized walnut protein isolate dispersion; (3) After the homogenization is completed, 5% to 35% of pectin of walnut protein isolate is added, the mixture is stirred evenly, the pH value of the reaction system is adjusted to 3 to 8, the reaction is carried out at 50 to 80° C. for 60 to 480 min, and after the reaction is completed, the mixture is cooled to room temperature to obtain a walnut protein isolate-pectin covalent complex solution; (4) The walnut protein isolate-pectin covalent complex solution is subjected to vacuum freeze drying at a drying temperature of -65 to -72°C, a vacuum degree of 5 to 10 Pa, and a drying time of 24 to 48 h. After drying, the solution is ultrafinely ground and passed through a 120 to 140 mesh sieve to obtain a walnut protein isolate-pectin covalent complex powder.

9. A soluble plant-based walnut high-protein nutritional paste, characterized in that: Contains the walnut protein isolate-pectin covalent complex according to any one of claims 1 to 6, and erythritol, wherein the mass ratio of the walnut protein isolate-pectin covalent complex to erythritol is (4-10):

1.

10. The easily soluble plant-based walnut high-protein nutritional paste according to claim 9, characterized in that: Also contains calcium gluconate and vitamin D3.

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