Method for improving protein solubility of walnut meal through combination of ultrasonic-assisted restrictive enzymolysis and glycosylation modification

Abstract

The invention relates to a method for improving the protein solubility of walnut meal by combining ultrasonic-assisted restrictive enzymolysis with glycosylation modification, which comprises the following steps: degreasing walnut meal to obtain walnut degreased powder; the method comprises the following steps: dissolving walnut defatted powder by using deionized water, treating the walnut defatted powder by using double-frequency ultrasonic waves, magnetically stirring, centrifuging, taking supernatant, stirring, centrifuging, taking precipitate, and carrying out vacuum freeze drying to obtain walnut meal protein; carrying out restrictive enzymolysis on the walnut meal protein by using trypsin, and inactivating protease contained in enzymatic hydrolysate after enzymolysis; and adding the inactivated enzymatic hydrolysate into maltodextrin for a glycosylation reaction, after the glycosylation reaction, collecting a solution, freeze-drying to obtain a glycoprotein conjugate, and storing under a drying condition. According to the method disclosed by the invention, ultrasonic-assisted restrictive enzymolysis is combined with glycosylation modification to improve the protein solubility of the walnut meal, so that the reaction steps can be optimized, the reaction time is saved, the protein solubility is greatly improved, and the deep processing and comprehensive utilization of the walnut industry are further promoted.

Description

Technical Field

[0001] The invention relates to the technical field of protein modification, and in particular to a method for improving the solubility of walnut meal protein by utilizing ultrasound-assisted restricted enzymatic hydrolysis combined with glycosylation modification. Background Art

[0002] With the explosion of the world's population, sustainable plant protein replacing animal protein has become an inevitable development trend in the food industry. Traditional plant proteins such as soy protein have been developed and utilized to a great extent due to their good processing characteristics. The walnut meal left after oil extraction is also a high-quality plant protein resource. It contains about 54% protein, rich in amino acids, and has a balanced ratio of essential amino acids for the human body. It has many advantages in nutrition and health. In addition, the planting scale and output of walnuts are also expanding. Therefore, the in-depth utilization of walnut meal protein and the development of related products have attracted the attention of many food manufacturers.

[0003] However, walnut meal protein is an alkali-soluble hydrophobic protein with the highest hydrophobic amino acid ratio of 39.21%, and its main component is gluten (70%). Therefore, its solubility under neutral conditions is lower than that of other plant proteins. Low solubility usually brings many disadvantages to food, such as dry and rough taste, liquid state that is easy to precipitate, etc., and other functional properties such as thickening and foaming are limited by low solubility. In summary, improving the solubility of walnut meal protein is a huge challenge and a key issue that needs to be solved urgently.

[0004] In the past decade, various strategies have been developed to improve protein solubility, which can be generally divided into physical, chemical and enzymatic means according to the different technologies involved. However, a single means usually has its own limitations and disadvantages. The current trend is to combine different means to develop a hybrid method with synergistic effects. For example, a Chinese patent with publication number CN 114317660 B discloses a method for improving the utilization rate of walnut protein. Specifically, walnut peptides of >30KDa are prepared by enzymatic hydrolysis and ultrafiltration, and the walnut peptides are modified by glycosylation, and the various functional properties of the glycosylated walnut peptides obtained are improved. However, the reaction conditions of this method are complicated, the reaction process is as long as 47 hours, and only walnut polypeptides of >30KDa are utilized, which not only leads to excessively expensive production costs but also low reproducibility of the technology. The technology of combined modification by multiple means usually faces the problems of cumbersome steps, long time consumption, excessive protein loss in the reaction process, and high overall cost. This is also a problem that needs to be focused on in the current field of protein modification. Summary of the invention

[0005] Technical problems solved: In view of the technical problems existing in the prior art, such as cumbersome steps, long time consumption, excessive protein loss in the reaction process, and high overall cost, the present invention proposes a method for improving the solubility of walnut meal protein by combining ultrasound-assisted restriction enzymatic hydrolysis with glycosylation modification, which makes full use of the synergistic effect between different technologies, optimizes the reaction steps, and saves reaction time. Moreover, compared with a single modification method, the combined modification has the advantage of more obvious improvement in protein solubility.

[0006] Technical solution: A method for improving the solubility of walnut meal protein by using ultrasound-assisted restricted enzymatic hydrolysis combined with glycosylation modification, the steps are as follows: Step 1: Defatting walnut meal: Defatting the walnut meal, sieving it, obtaining defatted walnut powder and storing it for later use; Step 2, extracting walnut meal protein: using deionized water to dissolve walnut defatted powder, using dual-frequency ultrasound to treat the walnut defatted powder, centrifuging after magnetic stirring, taking the supernatant, stirring and centrifuging, taking the precipitate, and vacuum freeze-drying to obtain walnut meal protein; Step 3: restriction enzymolysis, using trypsin to perform restriction enzymolysis on the walnut meal protein obtained in step 2, and inactivating the protease contained in the enzymolysis solution after enzymolysis; Step 4: glycosylation reaction. Add the inactivated enzymatic hydrolysate to maltodextrin for glycosylation reaction. After the glycosylation reaction, collect the solution, freeze-dry it to obtain a glycoprotein conjugate, and store it under dry conditions.

[0007] Preferably, the defatting treatment in step 1 is as follows: walnut meal is sieved through a 180-mesh sieve, n-hexane is added, the solid-liquid ratio of walnut meal to n-hexane is 1:5, w / v, stirring at room temperature for 2-3 hours, standing for stratification, pouring out the n-hexane, repeating three times until the supernatant is clear, collecting the filter residue, evaporating it in a fume hood, and crushing it; after the defatting treatment, sieve it through an 80-mesh sieve to obtain the defatted walnut powder, which is then stored at -4°C for later use.

[0008] Preferably, in step 2, 22 kHz and 28 kHz dual-frequency ultrasound is used to treat the defatted walnut powder, the pH is adjusted to 10-11, magnetic stirring is performed and then centrifuged, the supernatant is taken, the pH is adjusted to 4-5, stirring is performed and then centrifuged, the precipitate is taken, the pH is adjusted to neutral, and vacuum freeze-dried.

[0009] Preferably, in step 2, the ultrasonic treatment power is 300 W and the ultrasonic time is 15 min.

[0010] Preferably, in step 2, the solid-liquid ratio of the defatted walnut powder to deionized water is 1:25, w / v.

[0011] Preferably, the restriction enzyme hydrolysis conditions in step three are as follows: deionized water is used to dissolve the protein, protein: water = 1:50, w / v, and the amount of trypsin added to each gram of protein is 5000 u.

[0012] Preferably, the hydrolysis time of the restriction enzyme hydrolysis in step three is 30-150 min, the hydrolysis temperature is 37° C., and the hydrolysis pH is 8.

[0013] Preferably, in step 4, the saccharification reaction time is 20-120 min, the temperature is 50-90° C., the pH is 6-11, and the mass ratio of maltodextrin to walnut meal protein is (1-3): (3-1).

[0014] Preferably, in step 4, the glycosylation reaction time is 20-40 min, the temperature is 60-90° C., and the pH is 9-11.

[0015] Preferably, in step 4, the mass ratio of maltodextrin to walnut meal protein is 1:2.

[0016] The present invention can not only increase the yield and purity of protein through dual-frequency ultrasonic treatment, but also promote the unfolding of protein structure, expose more protease binding sites, and accelerate the enzymatic reaction time. After restricted enzymatic hydrolysis, the protein part of the peptide chain is broken, the exposure of the ε-amino or N-terminal of the protein side chain increases, it is easier to bind to the carboxyl end of the carbohydrate, the degree of grafting between the protein and the sugar is deepened, and the glycosylation reaction time can be accelerated, and the generation of complex products in the glycosylation reaction can be reduced. This study has obtained a process for improving the solubility of mature walnut meal protein. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. Beneficial Effects

[0017] (1) The present invention can improve the yield and purity of the extracted protein by treating the defatted walnut powder with dual-frequency ultrasound, promote the unfolding of the protein structure, expose more protease binding sites, and accelerate the enzymatic reaction time; (2) The present invention uses trypsin to hydrolyze walnut meal protein in a limited manner. Trypsin can only induce hydrolysis of the carboxyl terminal peptide bonds of lysine and arginine without destroying the hydrophobic groups of amino acids, thereby providing good surface activity. This special cleavage method of trypsin improves the solubility and interfacial properties of walnut meal protein. Limited enzymatic hydrolysis can improve the function of protein and avoid the production of bitter peptides. Excessive hydrolysis can reduce the molecular weight of protein or may cause the protein to be completely digested by the enzyme, resulting in loss of protein function. (3) The present invention proposes a restricted enzymatic hydrolysis and combines it with glycosylation modification. The enzymatic hydrolysis reaction can increase the degree of grafting between protein and sugar, and at the same time can accelerate the glycosylation reaction time and reduce the generation of complex products in the glycosylation reaction, thus playing a synergistic effect. After enzymatic hydrolysis, the solubility of walnut meal protein increased by 46.44%. After the walnut meal protein was subjected to a glycosylation reaction, the solubility of the protein increased by another 8.56%. Finally, the solubility of the modified walnut meal protein was as high as 92.06%. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The solubility and grafting degree of the glycosylation product at different hydrolysis times; Figure 2 is the solubility diagram of different proteases; Figure 3 The solubility and grafting degree of glycosylation products under different sugar types; Figure 4 The solubility and grafting degree of glycosylation products under different sugar:protein ratio conditions are shown; Figure 5 The solubility and grafting degree of glycosylation products under different reaction pH conditions; Figure 6 The solubility and grafting degree of the glycosylation product under different reaction time conditions; Figure 7 The solubility and grafting degree of glycosylation products under different reaction temperature conditions; Figure 8 The solubility diagram of glycosylation products under different treatments; Fig. 9 This is a graph showing the protein yield after walnut meal protein was treated with ultrasound at different frequencies. DETAILED DESCRIPTION

[0019] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments.

[0020] The raw materials used in the examples of this specification, unless otherwise specified, are from common commercially available products, including: Walnut meal was purchased from Kashgar Guanghua Modern Agriculture Co., Ltd. Example 1

[0021] This embodiment provides a method for improving the solubility of walnut meal protein by using ultrasound-assisted restricted enzymatic hydrolysis combined with glycosylation modification, and the specific steps are as follows: 1. Preparation of defatted walnut powder: The walnut meal was sieved through a 180-mesh sieve, and n-hexane was added (the solid-liquid ratio of walnut meal to n-hexane (w / v) was 1:5), stirred for 3 hours, allowed to stand for stratification, and the n-hexane was poured out (recovered by a rotary evaporator), and the process was repeated three times until the supernatant was clear. The filter residue was collected and evaporated in a decanter, crushed, sieved through an 80-mesh sieve, and stored at -4°C to obtain walnut defatted powder.

[0022] 2. Walnut meal protein extraction: Take 100 g of defatted walnut powder, add deionized water (the solid-liquid ratio of defatted walnut powder to deionized water (w / v) is 1:25), use 22 kHz and 28 kHz dual-frequency ultrasound to treat the defatted walnut powder, where the ultrasonic power is 300 w, the ultrasonic time is 15 min, after ultrasonic treatment, adjust the pH to 10, magnetic stirring for 1.5 hours, and then centrifuge at 7000 r / min for 10 min. Take the supernatant, adjust the pH to 4.5, stir for 1.5 hours, centrifuge at 7000 r / min for 15 min, take the precipitate, adjust the pH to 7, and vacuum freeze-dry to obtain walnut meal protein.

[0023] See also Fig. 9 The applicant used the single variable method to change the frequency of ultrasonic treatment (22 kHZ, 28 kHZ, 40 kHZ, 28+22 kHZ, 22+40 kHZ and 22+28+40 kHZ). From the results in the figure, it can be seen that the protein yield under 28+22 kHZ conditions is relatively high.

[0024] 3. Determination of basic ingredients 3.1 Determination of protein content With reference to the national standard "GB 5009.5-2016" for the determination of protein in food, the purity of walnut meal protein was measured to be 80.02%.

[0025] 3.2 Determination of fat content Referring to the national standard "GB 5009.6-2016" for the determination of fat in food, the residual fat was measured to be 2.7%.

[0026] 3.3 Determination of ash content With reference to the national standard “GB 5009.4-2016” for the determination of ash in food, the ash content was measured to be 3.2%.

[0027] 3.4 Determination of moisture content Referring to the national standard "GB 5009.3-2016" Determination of moisture in food, the moisture content was measured to be 8.3%.

[0028] 4. Restriction enzymatic hydrolysis and glycosylation reaction The walnut meal protein prepared in step 2 was subjected to restricted enzymatic hydrolysis. Deionized water was used to dissolve the walnut meal protein, protein: water = 1: 50 (w / v), and trypsin was used for restricted enzymatic hydrolysis. The amount of protease added per gram of protein was 5000u, the hydrolysis time was 1 h, the hydrolysis temperature was 37°C, the hydrolysis pH was 8, and the protease contained in the enzymatic hydrolysis solution was inactivated after enzymatic hydrolysis.

[0029] The inactivated enzymatic hydrolysate was added to maltodextrin for glycosylation reaction. The glycosylation reaction time was 1 h, the reaction temperature was 85°C, the pH was 10, and the mass ratio of maltodextrin to walnut meal protein was 1:1. After the glycosylation reaction, the solution was collected and freeze-dried to obtain a glycoprotein conjugate, which was stored under dry conditions. Example 2

[0030] Same as Example 1, except that the hydrolysis time of the restriction enzyme hydrolysis in step 4 was adjusted to 30 min, 60 min, 90 min, 120 min and 150 min, the solubility of the obtained glycosylation reaction product was determined by the Coomassie Brilliant Blue method, and the grafting degree of the protein and polysaccharide after the reaction was determined by the OPA method. Figure 1 ,From the figure, it can be seen that the best hydrolysis time is 30 min, specifically 81.07%. Example 3

[0031] Same as Example 1, except that the hydrolysis time of the restriction enzymatic hydrolysis was adjusted to 30 min, the enzymes of the restriction enzymatic hydrolysis were adjusted to trypsin, alkaline protease, flavor protease and papain, respectively, and the solubility of the obtained glycosylation reaction product was determined by the Coomassie brilliant blue method. The results are shown in Figure 2 As can be seen from the figure, trypsin has the highest solubility. This is because trypsin can only induce hydrolysis of the carboxyl terminal peptide bonds of lysine and arginine without destroying the hydrophobic groups of amino acids, thus providing good surface activity.

[0032] At the same time, due to the special cutting method of trypsin, the solubility and interfacial properties of walnut meal protein are improved. Limited enzymatic hydrolysis can improve the function of protein and avoid the production of bitter peptides. Excessive hydrolysis will reduce the molecular weight of protein or may cause the protein to be completely digested by the enzyme, resulting in loss of protein function. Example 4

[0033] Same as Example 1, except that the hydrolysis time of the restricted enzymatic hydrolysis was adjusted to 30 min, maltodextrin was replaced by dextran, high-fat pectin, sodium alginate or chitosan, the solubility of the obtained glycosylated product was determined by the Coomassie brilliant blue method, and the grafting degree of the protein and polysaccharide after the reaction was determined by the OPA method. Figure 3 ,As can be seen from the figure, the polysaccharide with the highest solubility is maltodextrin. Example 5

[0034] Same as Example 1, except that the hydrolysis time of the restricted enzymatic hydrolysis was adjusted to 30 min, the mass ratio of maltodextrin to walnut meal protein was adjusted to 1:3, 1:2, 1:1, 2:1 and 3:1 respectively, the solubility of the glycosylated product was determined by the Coomassie brilliant blue method, and the grafting degree of the protein and polysaccharide after the reaction was determined by the OPA method. Figure 4 ,As can be seen from the figure, the ratio with the highest solubility is 1:2, specifically 86.53%. Example 6

[0035] The same as Example 1, except that the hydrolysis time of the restricted enzymatic hydrolysis was adjusted to 30 min, the mass ratio of maltodextrin to walnut meal protein was adjusted to 1:2, the pH of the glycosylation reaction was adjusted to 6, 7, 8, 9 or 10, the solubility of the obtained glycosylated product was determined by the Coomassie brilliant blue method, and the grafting degree of the protein and the polysaccharide after the reaction was determined by the OPA method. Figure 5 ,As can be seen from the figure, the pH with the highest solubility is 9, specifically 87.62%. Example 7

[0036] The same as Example 1, except that the hydrolysis time of the restricted enzymatic hydrolysis was adjusted to 30 min, the mass ratio of maltodextrin to walnut meal protein was adjusted to 1:2, the pH of the glycosylation reaction was adjusted to 9, the glycosylation reaction time was adjusted to 20 min, 40 min, 60 min, 80 min and 100 min, respectively, the solubility of the obtained glycosylation reaction product was determined by the Coomassie brilliant blue method, and the grafting degree of the protein and the polysaccharide after the reaction was determined by the OPA method. Results are shown in Figure 6 ,From the figure, it can be seen that the reaction time with the highest solubility is 40 min, specifically 91.78%. Example 8

[0037] The same as Example 1, except that the hydrolysis time of the restricted enzymatic hydrolysis was adjusted to 30 min, the mass ratio of maltodextrin to walnut meal protein was adjusted to 1:2, the glycosylation reaction pH was adjusted to 9, the glycosylation reaction time was adjusted to 30 min, the glycosylation reaction temperature was adjusted to 50°C, 60°C, 70°C, 80°C or 90°C, the solubility of the obtained glycosylation reaction product was determined by the Coomassie Brilliant Blue method, and the grafting degree of the protein and polysaccharide after the reaction was determined by the OPA method. Results are shown in Figure 7 ,From the figure, it can be seen that the reaction temperature with the highest solubility is 90 ℃, specifically 91.46%. Example 9

[0038] The same as Example 1, except that the hydrolysis time of the restricted enzymatic hydrolysis is adjusted to 30 min, the mass ratio of maltodextrin to walnut meal protein is adjusted to 1:2, the glycosylation reaction pH is adjusted to 9, the glycosylation reaction time is adjusted to 40 min, the glycosylation reaction temperature is adjusted to 90°C, the solubility of the obtained glycosylation reaction product is determined by the Coomassie Brilliant Blue method, and the degree of grafting of the protein and the polysaccharide after the reaction is determined by the OPA method. Comparative Example 1

[0039] The difference is the same as Example 9, in which the conventional alkali dissolution and acid precipitation method is used to extract walnut meal protein. The specific steps are as follows: 1. Preparation of defatted walnut powder: The walnut meal was sieved through a 180-mesh sieve, and n-hexane was added (the solid-liquid ratio of walnut meal to n-hexane (w / v) was 1:5), stirred for 3 hours, allowed to stand for stratification, and the n-hexane was poured out (recovered by a rotary evaporator), and the process was repeated three times until the supernatant was clear. The filter residue was collected and evaporated in a decanter, crushed, sieved through an 80-mesh sieve, and stored at -4°C to obtain walnut defatted powder.

[0040] 2. Walnut meal protein extraction: Take 100 g of defatted walnut powder, add deionized water (the solid-liquid ratio of defatted walnut powder to deionized water (w / v) is 1:25), adjust the pH to 10, stir magnetically for 1.5 hours, and then centrifuge at 7000 r / min for 10 min. Take the supernatant, adjust the pH to 4.5, stir for 1.5 hours, centrifuge at 7000 r / min for 15 min, take the precipitate, adjust the pH to 7, and vacuum freeze-dry to obtain the product walnut meal protein control. Comparative Example 2

[0041] The same as Example 9, except that this comparative example uses ultrasound-assisted extraction of walnut meal protein, and the specific steps are as follows: 1. Preparation of defatted walnut powder: The walnut meal was sieved through a 180-mesh sieve, and n-hexane was added (the solid-liquid ratio of walnut meal to n-hexane (w / v) was 1:5), stirred for 3 hours, allowed to stand for stratification, and the n-hexane was poured out (recovered by a rotary evaporator), and the process was repeated three times until the supernatant was clear. The filter residue was collected and evaporated in a decanter, crushed, sieved through an 80-mesh sieve, and stored at -4°C to obtain walnut defatted powder.

[0042] 2. Walnut meal protein extraction: Take 100 g of defatted walnut powder, add deionized water (the solid-liquid ratio (w / v) of defatted walnut powder to deionized water is 1:25), use 22 kHz and 28 kHz dual-frequency ultrasound to treat the defatted walnut powder, where the ultrasonic power is 300 w, the ultrasonic time is 15 min, after ultrasonic treatment, adjust the pH to 10, magnetic stirring for 1.5 hours, and then centrifuge at 7000 r / min for 10 min. Take the supernatant, adjust the pH to 4.5, stir for 1.5 hours, centrifuge at 7000 r / min for 15 min, take the precipitate, adjust the pH to 7, and vacuum freeze-dry to obtain the product walnut meal protein US. Comparative Example 3

[0043] The difference is the same as Example 9, in which the walnut meal protein is extracted by ultrasound-assisted alkali dissolution and acid precipitation method and subjected to restricted hydrolysis modification treatment, as follows: 1. Preparation of defatted walnut powder: The walnut meal was sieved through a 180-mesh sieve, and n-hexane was added (the solid-liquid ratio of walnut meal to n-hexane (w / v) was 1:5), stirred for 3 hours, allowed to stand for stratification, and the n-hexane was poured out (recovered by a rotary evaporator), and the process was repeated three times until the supernatant was clear. The filter residue was collected and evaporated in a decanter, crushed, sieved through an 80-mesh sieve, and stored at -4°C to obtain walnut defatted powder.

[0044] 2. Walnut meal protein extraction: Take 100 g of defatted walnut powder, add deionized water (the solid-liquid ratio of defatted walnut powder to deionized water (w / v) is 1:25), use 22 kHz and 28 kHz dual-frequency ultrasound to treat the defatted walnut powder, where the ultrasonic power is 300 w, the ultrasonic time is 15 min, after ultrasonic treatment, adjust the pH to 10, magnetic stirring for 1.5 hours, and then centrifuge at 7000 r / min for 10 min. Take the supernatant, adjust the pH to 4.5, stir for 1.5 hours, centrifuge at 7000 r / min for 15 min, take the precipitate, adjust the pH to 7, and vacuum freeze-dry to obtain walnut meal protein.

[0045] 3. Restricted hydrolysis modification treatment The walnut meal protein prepared in step 2 is subjected to restricted enzymolysis, deionized water is used to dissolve the walnut meal protein, protein: water = 1: 50 (w / v), trypsin is used for restricted enzymolysis, the amount of protease added to each gram of protein is 5000u, the hydrolysis time is 30min, the hydrolysis temperature is 37°C, the hydrolysis pH is 8, and the protease contained in the enzymolysis solution is inactivated after enzymolysis. The solution is collected, freeze-dried to obtain the product walnut meal protein USH, and stored under dry conditions. Comparative Example 4

[0046] The difference is the same as Example 9, in which the walnut meal protein is extracted by ultrasound-assisted alkali dissolution and acid precipitation method and the saccharification reaction is directly carried out without restrictive hydrolysis modification treatment, as follows: 1. Preparation of defatted walnut powder: The walnut meal was sieved through a 180-mesh sieve, and n-hexane was added (the solid-liquid ratio of walnut meal to n-hexane (w / v) was 1:5), stirred for 3 hours, allowed to stand for stratification, and the n-hexane was poured out (recovered by a rotary evaporator), and the process was repeated three times until the supernatant was clear. The filter residue was collected and evaporated in a decanter, crushed, sieved through an 80-mesh sieve, and stored at -4°C to obtain walnut defatted powder.

[0047] 2. Walnut meal protein extraction: Take 100 g of defatted walnut powder, add deionized water (the solid-liquid ratio of defatted walnut powder to deionized water (w / v) is 1:25), use 22 kHz and 28 kHz dual-frequency ultrasound to treat the defatted walnut powder, where the ultrasonic power is 300 w, the ultrasonic time is 15 min, after ultrasonic treatment, adjust the pH to 10, magnetic stirring for 1.5 hours, and then centrifuge at 7000 r / min for 10 min. Take the supernatant, adjust the pH to 4.5, stir for 1.5 hours, centrifuge at 7000 r / min for 15 min, take the precipitate, adjust the pH to 7, and vacuum freeze-dry to obtain walnut meal protein.

[0048] 3. Glycosylation reaction: Deionized water was used to dissolve walnut meal protein, protein: water = 1:50 (w / v), and maltodextrin was added for saccharification reaction. The saccharification reaction time was 40 min, the reaction temperature was 90℃, the pH was 9, and the mass ratio of maltodextrin to walnut meal protein was 1:2. After the saccharification reaction, the solution was collected and freeze-dried to obtain the product walnut meal protein USG, which was stored under dry conditions. Comparative Example 5

[0049] The same as Example 9, except that, in this comparative example, the mass ratios of maltodextrin and walnut meal protein are adjusted to 1:1, 1:2 and 1:3, respectively, to obtain products USHG-1, USHG-2 and USHG-3.

[0050] The solubility of the products prepared in the above comparative examples 1-5 was measured. For specific results, see Figure 8, where Control represents the traditional alkali dissolution and acid precipitation method for extracting walnut meal protein; US represents ultrasound-assisted extraction of walnut meal protein; USH represents ultrasound-assisted alkali dissolution and acid precipitation method for extracting walnut meal protein and undergoing restricted hydrolysis modification; USG represents ultrasound-assisted alkali dissolution and acid precipitation method for extracting walnut meal protein and directly undergoing glycosylation without restricted hydrolysis modification; USHG-1 represents ultrasound-assisted alkali dissolution and acid precipitation method for extracting walnut meal protein and undergoing restricted hydrolysis modification for coordinated glycosylation, with a polysaccharide to protein concentration ratio of 1:1; USHG-2 represents ultrasound-assisted alkali dissolution and acid precipitation method for extracting walnut meal protein and undergoing restricted hydrolysis modification for coordinated glycosylation, with a polysaccharide to protein concentration ratio of 1:2; USHG-3 represents ultrasound-assisted alkali dissolution and acid precipitation method for extracting walnut meal protein and undergoing restricted hydrolysis modification for coordinated glycosylation, with a polysaccharide to protein concentration ratio of 1:3. From Figure 8 It can be seen that USHG-2, which is Example 9 of this specification, has the best result, reaching 92.06%.

[0051] This is because the cavitation effect of ultrasound destroys the hydrogen bonds between protein molecules, which reduces the size of aggregates and increases the solubility of proteins. After the protein is hydrolyzed by restricted hydrolysis, some peptide chains are interrupted, the polar water environment changes, and the solubility is significantly increased. The addition of maltodextrin introduces hydrophilic groups into the protein system, further increasing the solubility. The results of USG show that it is necessary to perform enzymatic hydrolysis for 30 minutes before glycosylation.

[0052] Finally, it should be noted that the above implementation modes are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned implementation modes, a person of ordinary skill in the art should understand that the technical solutions described in the aforementioned implementation modes can still be modified, or some or all of the technical features therein can be replaced by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the implementation modes of the present invention.

Claims

1. A method for improving the solubility of walnut meal protein by using ultrasound-assisted restricted enzymatic hydrolysis combined with glycosylation modification, characterized in that: Here are the steps: Step 1: Defatting walnut meal: Defatting the walnut meal, sieving it, obtaining defatted walnut powder and storing it for later use; Step 2, extracting walnut meal protein: using deionized water to dissolve walnut defatted powder, using dual-frequency ultrasound to treat the walnut defatted powder, centrifuging after magnetic stirring, taking the supernatant, stirring and centrifuging, taking the precipitate, and vacuum freeze-drying to obtain walnut meal protein; Step 3: restriction enzymolysis, using trypsin to perform restriction enzymolysis on the walnut meal protein obtained in step 2, and inactivating the protease contained in the enzymolysis solution after enzymolysis; Step 4: glycosylation reaction. Add the inactivated enzymatic hydrolysate to maltodextrin for glycosylation reaction. After the glycosylation reaction, collect the solution, freeze-dry it to obtain a glycoprotein conjugate, and store it under dry conditions.

2. The method of improving the solubility of walnut meal protein by combining ultrasound-assisted restricted enzymolysis with glycosylation modification according to claim 1, characterized in that: The defatting treatment in step 1 is specifically as follows: walnut meal is sieved through a 180-mesh sieve, n-hexane is added, the solid-liquid ratio of walnut meal to n-hexane is 1:5, w / v, stirring at room temperature for 2-3 hours, standing for stratification, pouring out the n-hexane, repeating three times until the supernatant is clear, collecting the filter residue and evaporating it in a fume hood, and crushing it; after the defatting treatment, sieve through an 80-mesh sieve to obtain the defatted walnut powder, which is then stored at -4°C for later use.

3. The method of improving the solubility of walnut meal protein by combining ultrasound-assisted restricted enzymolysis with glycosylation modification according to claim 1, characterized in that: In the step 2, 22 kHz and 28 kHz dual-frequency ultrasound is used to treat the defatted walnut powder, the pH is adjusted to 10-11, magnetic stirring is performed and then centrifuged, the supernatant is taken, the pH is adjusted to 4-5, stirring is performed and then centrifuged, the precipitate is taken, the pH is adjusted to neutral, and vacuum freeze-dried.

4. The method of improving the solubility of walnut meal protein by combining ultrasound-assisted restricted enzymolysis with glycosylation modification according to claim 1, characterized in that: In the step 2, the ultrasonic treatment power is 300 W and the ultrasonic time is 15 min.

5. The method of improving the solubility of walnut meal protein by combining ultrasound-assisted restricted enzymolysis with glycosylation modification according to claim 1, characterized in that: In the step 2, the solid-liquid ratio of the defatted walnut powder to deionized water is 1:25, w / v.

6. The method of improving the solubility of walnut meal protein by using ultrasound-assisted restricted enzymolysis combined with glycosylation modification according to claim 1, characterized in that: The restrictive enzymatic hydrolysis conditions in step three are as follows: deionized water is used to dissolve walnut meal protein, protein: water = 1:50, w / v, and the amount of trypsin added to each gram of protein is 5000 u.

7. A method for improving the solubility of walnut meal protein by combining ultrasound-assisted restricted enzymolysis with glycosylation modification according to claim 1 or 6, characterized in that: The hydrolysis time of the restriction enzyme hydrolysis in step 3 is 30-150 min, the hydrolysis temperature is 37° C., and the hydrolysis pH is 8.

8. The method of improving the solubility of walnut meal protein by using ultrasound-assisted restricted enzymolysis combined with glycosylation modification according to claim 1, characterized in that: In the step 4, the saccharification reaction time is 20-120 min, the temperature is 50-90° C., the pH is 6-11, and the mass ratio of maltodextrin to walnut meal protein is (1-3): (3-1).

9. The method of improving the solubility of walnut meal protein by using ultrasound-assisted restricted enzymolysis combined with glycosylation modification according to claim 8, characterized in that: In the step 4, the glycosylation reaction time is 20-40 min, the temperature is 60-90° C., and the pH is 9-11.

10. The method of improving the solubility of walnut meal protein by using ultrasound-assisted restricted enzymatic hydrolysis combined with glycosylation modification according to claim 1, characterized in that: In the step 4, the mass ratio of maltodextrin to walnut meal protein is 1:2.

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