Preparation method of walnut protein isolate

Abstract

The application discloses a walnut protein isolate preparation method, and the walnut protein isolate prepared by the method has the effect of improving memory impairment of the elderly and can be used for preparing acid food, emulsified food and foamed food. The method uses defatted walnut powder as raw material, utilizes reverse micelle dissolution, isoelectric point precipitation and the high solubility principle of sodium bis(2-ethylhexyl) succinate sulfonate in ethanol to prepare high-quality walnut protein isolate. The walnut protein isolate prepared by the method has the advantages of high-quality apparent properties, high protein content, strong solubility, strong emulsifying capacity, strong foaming capacity, high in-vitro old model digestion rate, strong AChE inhibiting capacity of digestion products and strong antioxidant capacity.

Description

Technical Field

[0001] This invention belongs to the field of functional foods, specifically relating to a method for preparing walnut protein isolate. Background Technology

[0002] With the aging population, the number of people suffering from neurocognitive disorders such as memory impairment has increased significantly, with the elderly accounting for the largest proportion. Memory impairment is defined as a pathological state characterized by a persistent decline in function in one or more cognitive processes, including recall, storage, retention, and retrieval. It is a core symptom of neurodegenerative diseases such as Alzheimer's disease, vascular dementia, and Lewy body dementia. This condition leads to the progressive loss of patients' daily living abilities, placing a dual burden on family care systems and social healthcare. Therefore, building a precise intervention and prevention system has become an urgent issue with significant public health value and socioeconomic benefits.

[0003] Walnut meal, a byproduct of walnut oil extraction, is not only high in protein (over 40%) but also possesses a unique amino acid composition (rich in arginine and with a balanced ratio of essential amino acids), highly compatible with the needs of neurotransmitter synthesis. Therefore, it is an excellent raw material source for developing memory-enhancing agents. Thus, extracting walnut protein isolate from walnut meal using modern processing techniques is of great significance for developing functional foods with potential cognitive-improving functions.

[0004] Currently, the alkali-dissolving and acid-precipitating process is widely used in the preparation of walnut protein isolate due to its simplicity and low cost. However, alkaline conditions may induce peptide chain breakage and promote covalent modification reactions, including amino acid residue aggregation, β-elimination, and racemization. Furthermore, excessive alkali treatment can lead to abnormal protein conformational unfolding and alterations in specific amino acid residues (such as arginine, lysine, and serine). Notably, studies have shown that arginine derivatives have a good protective effect on nerve cells, and their functional integrity directly affects cognitive improvement. Therefore, traditional extraction processes may reduce the neuroprotective activity of walnut protein isolate.

[0005] In addition, this process may cause severe denaturation of the extracted walnut protein, resulting in poor solubility and processing characteristics of the final protein product. This not only affects its processing properties but also its gastrointestinal digestibility and reduces its bioavailability. Summary of the Invention

[0006] This invention discloses a method for preparing walnut protein isolate. The walnut protein isolate prepared by this method has the effect of improving memory impairment in the elderly and can be used to prepare acidic foods, emulsified foods, and foamed foods. The method uses defatted walnut powder (walnut meal) as raw material and utilizes reverse micelle dissolution, isoelectric point precipitation, and the high solubility of sodium di(2-ethylhexyl)succinate sulfonate (AOT) in ethanol to prepare high-quality walnut protein isolate. Specifically, AOT, n-hexane, and phosphate buffer are mixed in a certain proportion to prepare a reverse micelle solution, and defatted walnut powder is dissolved in the solution. After centrifugation, the supernatant is mixed with an equal volume of phosphate buffer and stirred. After centrifugation again and standing, the mixture forms a three-layer system. The interfacial layer and the lower aqueous phase are collected, mixed evenly, and the pH is adjusted to the isoelectric point of walnut protein to precipitate the protein. After further centrifugation, the precipitate and the upper suspended solids are collected and mixed. Ethanol-NaCl solvent is added to the mixture to separate the protein and AOT. After centrifugation, the precipitate is collected and mixed with a small amount of water to reconstitute the protein solution. Small molecule impurities are further removed by ultrafiltration. The protein solution is then freeze-dried to obtain walnut protein isolate.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A method for preparing walnut protein isolate includes the following steps:

[0009] (1) Dissolve sodium di(2-ethylhexyl)succinate sulfonate in n-hexane to prepare solution A. Then mix buffer containing 0.05-0.1 M KCl with solution A and stir to form reverse micelle solution. Then mix defatted walnut powder with reverse micelle solution, stir evenly, centrifuge, and collect the supernatant to obtain walnut protein extract 1.

[0010] The concentration of solution A in step (1) is preferably 0.18 M;

[0011] The preferred volume ratio of the buffer solution to solution A in step (1) is 1:(18-22);

[0012] In step (1), the defatted walnut powder and the reverse micelle solution are preferably mixed at a ratio of 1:(15-20) (w / v);

[0013] The buffer solution mentioned in step (1) is a phosphate buffer or a Tris-HCl buffer with a pH of 7.3 to 7.7;

[0014] The defatted walnut powder mentioned in step (1) is a powder with an oil content of less than 5% obtained by pressing and / or solvent extraction of walnut kernels and then crushing it. It is also known as walnut meal.

[0015] Step (1) Centrifugation can be performed using a tubular centrifuge or a disc centrifuge to ensure that no obvious precipitate appears after centrifugation at a centrifugal force of 5000×g for 5 min.

[0016] (2) Mix walnut protein extract 1 with a buffer solution containing 0.8-1.0 M KCl, stir well, centrifuge, let stand to separate the layers, collect the middle layer and the lower aqueous phase, mix them to obtain walnut protein extract 2.

[0017] The preferred volume ratio of walnut protein extract 1 to buffer solution in step (2) is 1:(1-2);

[0018] The buffer solution in step (2) is a phosphate buffer or a Tris-HCl buffer with a pH of 7.8 to 8.2;

[0019] The stirring conditions described in steps (1) and (2) are: temperature 35-45 ℃, frequency 180-200 rpm, and time 30-60 min.

[0020] (3) Adjust the pH of walnut protein extract 2 to 4.5, let it stand, centrifuge, collect the supernatant and precipitate, mix them to obtain walnut protein isolate 3; add anhydrous ethanol as solvent to 0.1-0.5 mM NaCl to make the final concentration reach 0.01-0.03 mM to prepare solution B; mix walnut protein isolate 3 with solution B, stir evenly, let it stand, centrifuge, collect the precipitate to obtain walnut protein isolate 4;

[0021] In step (3), the walnut protein isolate 3 is preferably mixed with solution B at a ratio of 1:(20-30) (w / v);

[0022] (4) Add deionized water to walnut protein isolate 4, stir well, and then separate it through an ultrafiltration membrane with a membrane flux of 10000 Da. Collect the retentate, freeze dry it, and obtain walnut protein isolate.

[0023] The walnut protein isolate prepared by the above method has a high arginine content, strong antioxidant and AChE inhibitory activity, and a high in vitro digestibility under a simulated elderly digestion model. It can be used to prepare medicines and health foods that help improve memory.

[0024] Furthermore, the walnut protein isolate obtained by the above method can be used to prepare medicines and health foods that help improve memory in the elderly.

[0025] The walnut protein isolate obtained by the above method has high water solubility under acidic conditions and can be used to prepare acidic foods, including nutritional and sports drinks, acidified sauces, etc.

[0026] The walnut protein isolate obtained by the above method has excellent emulsifying properties, excellent foaming ability and stability, and can be used to prepare emulsified foods and foamed foods.

[0027] The present invention has the following advantages and effects compared with the prior art:

[0028] 1. This invention uses reverse micelle extraction combined with acid precipitation and alcohol washing to extract, enrich and purify walnut protein isolate. Compared with traditional alkaline extraction, it can reduce the loss of amino acids and improve its processing characteristics. This method can be used for both industrial production and experimental theoretical research, and has outstanding innovation.

[0029] 2. The method of the present invention introduces an acid precipitation and alcohol washing process by adding non-toxic and harmless hydrochloric acid solution and ethanol-NaCl solution to the existing reverse micelle protein extraction process, which can reduce the residual surfactant in walnut protein isolate. The process is safe, efficient, green and environmentally friendly and sustainable.

[0030] 3. The walnut protein isolate prepared by this invention has the advantages of excellent apparent properties, high protein content, strong solubility, strong emulsifying ability, strong foaming ability, high digestibility in in vitro aging models, and strong AChE inhibition and antioxidant capacity of digestion products. Attached Figure Description

[0031] Figure 1 These are the apparent characteristics of walnut protein isolate;

[0032] Figure 2 It refers to the solubility of walnut protein isolate;

[0033] Figure 3 This refers to the residual AOT content of walnut protein isolate;

[0034] Figure 4 This refers to the emulsifying properties of walnut protein isolate;

[0035] Figure 5 This refers to the foaming properties of walnut protein isolate;

[0036] Figure 6 This refers to the digestibility of walnut protein isolate in an elderly in vitro digestion model;

[0037] Figure 7 The AChE inhibition rate of walnut protein isolate in an elderly in vitro digestion model;

[0038] Figure 8 The DPPH clearance rate of walnut protein isolate in an elderly in vitro digestion model;

[0039] Figure 9 The ABTS clearance rate of walnut protein isolate in an elderly in vitro digestion model;

[0040] Wherein, A: Walnut protein isolate A; B: Walnut protein isolate B; C: Walnut protein isolate C; D: Walnut protein isolate 1; E: Walnut protein isolate 2; F: Walnut protein isolate 3; different letters indicate significant differences between samples (p<0.05). Detailed Implementation

[0041] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0042] The experimental methods for the test indicators involved in the embodiments of the present invention are as follows:

[0043] 1. Walnut protein isolate composition test

[0044] (1) Protein purity determination: Take 2 g of walnut protein isolate and determine the protein content using the Kjeldahl method (refer to GB 5009.5-2016). The protein purity of walnut protein isolate is calculated according to equation (1):

[0045] Protein purity (%) = Protein content of walnut protein isolate / Mass of walnut protein isolate × 100 (1)

[0046] (2) Determination of total sugar content: Prepare a glucose standard stock solution and plot a standard curve using the phenol-sulfuric acid method. Take an appropriate amount of walnut protein isolate and perform three parallel experiments according to the standard curve operation method. After completion, measure the absorbance value at 595 nm and calculate the total sugar content of the walnut protein isolate.

[0047] (3) Ash content was determined according to GB 5009.4-2016, moisture content was determined according to GB 5009.3-2016, and fat content was determined according to GB 5009.6-2016.

[0048] 2. Measurement of colorimetry

[0049] The color of walnut protein isolate was measured using a portable precision colorimeter (model WSC-2B, Shanghai Instrument & Electronics Physical Optical Instrument Co., Ltd.).

[0050] 3. Measurement of AOT

[0051] Prepare a 0.007% methylene blue solution using a 1% Na₂SO₄ aqueous solution. Then, mix 1 mL of the methylene blue solution with 5 mL of chloroform. The methylene blue reacts with AOT to form a salt, which dissolves in the chloroform to form a colored layer. Remove the upper aqueous phase containing excess dye. Transfer the chloroform phase to a cuvette and measure its absorbance using a 650 nm UV spectrophotometer. Calculate the AOT content in the protein using a standard curve prepared with AOT.

[0052] 4. Method for determining the solubility of walnut protein isolate

[0053] Walnut protein isolate and ultrapure water were mixed at a ratio of 1:20 (w / v) to prepare a suspension. The pH of the suspension was adjusted to 2.0-12.0 using 0.1 M HCl or 0.1 M NaOH. After mixing the suspension, it was centrifuged (4000 g, 30 min, 25 ℃). The protein content of the supernatant was determined by the Kjeldahl method, and the solubility was calculated according to equation (2):

[0054] Solubility (%) = Protein content in supernatant / Protein content in walnut protein isolate sample × 100 (2)

[0055] 5. Determination of emulsifying ability

[0056] Emulsifying properties, including the emulsifying activity index (EAI) and the emulsifying stability index (ESI), were determined by turbidity method. At room temperature, 30 mL of protein solution (0.2%, w / v) was thoroughly mixed with 10 mL of soybean oil at 10000 × g for 1 minute using a homogenizer (model T25, IKA Instruments GmbH, Germany). At 0 and 10 minutes after homogenization, 40 μL of the emulsion was transferred from the bottom of the container and dispersed in 5 mL of 0.1% SDS (w / v). The absorbance at 500 nm was measured spectrophotometrically. EAI and ESI were calculated according to equations (3) and (4), respectively:

[0057] EAI (m 2 / g) = 2 × 2.303 / (c × φ (1-θ) × 10 4 ) × A0× Dilution factor (3)

[0058] ESI (%) = A 10 / A0× 100(4)

[0059] Where A0 and A 10 The absorbance values ​​are at 0 minutes and 10 minutes after emulsion homogenization, respectively. The dilution factor is 100, c is the protein concentration (g / mL), φ is the optical path (1 cm), and θ is the oil volume fraction (0.20).

[0060] 6. Determination of foaming ability / stability

[0061] Walnut protein isolate and ultrapure water were mixed at a ratio of 1:100 (w / v) to prepare a suspension (V1). After homogenization at 11520×g for 2 minutes, the foam volume (V2) and liquid volume (V3) were measured. The foam volume (V4) was measured again after the sample was allowed to stand for 30 minutes. The foaming capacity and foam stability were calculated using equations (5) and (6):

[0062] Foaming capacity (%) = (V2 + V3 - V1) / V1 × 100 (5)

[0063] Foam stability (%) = V4 / V2 × 100 (6)

[0064] 7. Determination of hydrolyzed amino acids

[0065] After the sample was hydrolyzed with 6 M hydrochloric acid at 110 °C for 24 hours, it was measured using a fully automated amino acid analyzer (model L-8900, Hitachi Instruments, Japan).

[0066] 8. Establishment of an in vitro digestive model of the elderly and determination of digestibility.

[0067] 3 g of walnut protein isolate was digested in 3 mL of simulated saliva at 7 rpm for 2 min. Then, 6 mL of simulated gastric juice (containing 1500 U / mL pepsin) was added, and the pH was adjusted to 4.5 with 5 mol / L HCl solution. The digestion was continued at 7 rpm for 180 min. Finally, 12 mL of simulated intestinal juice (containing 59.4 mg / mL ox bile salts, 46 U / mL trypsin, and 23 U / mL pancreatic lipase) was added, and the pH was adjusted to 6.6 with 1 mol / L NaOH. The digestion was continued at 7 rpm for 180 min. After digestion, all samples were treated at 90℃ for 10 min to inactivate the enzymes. After thawing, all digested solutions were centrifuged at 4℃ and 6000 r / min for 15 min. The protein content in the supernatant was further determined using a BCA protein kit, and the digestibility was calculated according to equation (7).

[0068] Digestibility (%) = Protein content in supernatant / Protein content of walnut protein isolate used for digestion × 100 (7)

[0069] 9. Determination of AChE inhibition rate

[0070] 30 μL ACh (7.5 mM), 125 μL DTNB (3 mM), 40 μL HEPES (pH 8.0, 50 mM, containing 0.1% bovine serum albumin), and 50 μL sample (10 mg / mL) were added to 96-well plates, mixed, and incubated at 37 °C for 15 min. After incubation, 30 μL of AChE (0.055 U / mL) was added to begin the assay. The assay was performed using a microplate reader. The wavelength was 412 nm, and the assay time was 15 min. The formula for calculating the inhibitory activity of the sample against AChE is shown in equation (8):

[0071] AChE inhibitory activity (%) = [1-(AChE)] 样品 -A 样品空白 / A 对照 -A 对照空白 )] ×100 (8)

[0072] Among them, A 样品 A represents the absorbance of the sample group. 样品空白 For the sample group without enzymes, A 对照 For the enzyme-added but sample-free group, A 对照空白 This is the sample group without enzymes or other additives.

[0073] 10. Determination of DPPH (2,2-biphenyl-1-picrylhydrazine) scavenging ability

[0074] Mix 2 mL of sample solution with 2.0 mL of DPPH solution (0.2 mM). Then vortex the mixture and keep it in the dark for 30 minutes. Measure the absorbance at 517 nm using spectrophotometry. Calculate the DPPH radical scavenging activity of each protein sample using equation (9):

[0075] DPPH free radical scavenging activity (%) = [1-(A 样品 -A 空白 )] / A 对照 ×100 (9)

[0076] Where A 样品 A 对照 and A 空白 These are the absorbance values ​​for the protein sample, control, and blank, respectively.

[0077] 11. Determination of ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) scavenging ability

[0078] The absorbance of ABTS (7 mM ABTS and 2.45 mM potassium persulfate) at 734 nm was adjusted to 0.70 ± 0.02 using 75 mM phosphate buffer (pH 7.4). Then, 50 μL of sample solution was added to 150 μL of diluted ABTS solution, and ABTS· scavenging activity was determined. The ABTS radical scavenging activity of each protein sample was calculated using equation (10):

[0079] ABTS free radical scavenging activity (%) = [1-(A 样品 -A 空白 )] / A 空白 ×100 (10)

[0080] Where A 样品 It is the absorbance of the sample, A 空白 It is the absorbance of the blank.

[0081] Example 1

[0082] A method for preparing walnut protein isolate includes the following steps:

[0083] (1) Dissolve sodium di(2-ethylhexyl)succinate sulfonate in n-hexane to prepare a solution A with a concentration of 0.18 M. Then mix 50 mM phosphate buffer (containing 0.05 MKCl, pH=7.3) with solution A at a ratio of 1:18 (v / v) and stir at 30 r / min for 30 min to form reverse micelles; then mix defatted walnut powder with the reverse micelles at a ratio of 1 g:15 mL (w / v), stir evenly, centrifuge, and collect the supernatant to obtain walnut protein extract A;

[0084] (2) Mix walnut protein extract A with an equal volume of 50 mM phosphate buffer (containing 1.0 MKCl, pH=7.8), stir well, centrifuge, let stand for 10 min, collect the middle layer and the lower aqueous phase, mix them, and obtain walnut protein extract A.

[0085] (3) Adjust the pH of walnut protein extract A to 4.5, let it stand for 1 h, centrifuge, collect the supernatant and precipitate, mix them to obtain walnut protein isolate A; add anhydrous ethanol as solvent to 0.1 mM NaCl to make the final concentration 0.01 mM to prepare solution B; mix walnut protein isolate A and solution B at a ratio of 1 g: 20 mL (w / v), stir evenly, let it stand for 1 h, centrifuge, collect the precipitate to obtain walnut protein isolate A;

[0086] (4) Add 10 times the mass of deionized water to walnut protein isolate A, stir well, and then separate it through an ultrafiltration membrane with a membrane flux of 10000 Da. Collect the retentate, freeze dry it, and obtain walnut protein isolate A.

[0087] The basic components of walnut protein isolate A are shown in Table 1.

[0088] The amino acid composition of walnut protein isolate A is shown in Table 2.

[0089] The phenotypic properties of walnut protein isolate A are shown in [reference needed]. Figure 1 .

[0090] The solubility of walnut protein isolate A is shown in the figure. Figure 2 .

[0091] For the foaming properties / stability of walnut protein isolate A, see [link to relevant documentation]. Figure 3 .

[0092] The emulsifying properties of walnut protein isolate A are shown in [link to documentation]. Figure 4 .

[0093] The foaming properties of walnut protein isolate A are shown in [link to relevant documentation]. Figure 5 .

[0094] The digestibility of walnut protein isolate A in an elderly in vitro digestion model is shown in the figure. Figure 6 .

[0095] The AChE inhibition rate of walnut protein isolate A in an elderly in vitro digestion model is shown in the figure. Figure 7 .

[0096] The DPPH clearance rate of walnut protein isolate A in an elderly in vitro digestive model is shown in the figure. Figure 8 .

[0097] The ABTS clearance rate of walnut protein isolate A in an elderly in vitro digestion model is shown in the figure. Figure 9 .

[0098] Example 2

[0099] A method for preparing walnut protein isolate includes the following steps:

[0100] (1) Dissolve sodium di(2-ethylhexyl)succinate sulfonate in n-hexane to prepare a solution A with a concentration of 0.18 M. Then mix 50 mM phosphate buffer (containing 0.075 MKCl, pH=7.5) with solution A at a ratio of 1:20 (v / v) and stir at 40 r / min for 30 min to form reverse micelles; then mix defatted walnut powder with the reverse micelles at a ratio of 1 g:18 mL (w / v), stir evenly, centrifuge, and collect the supernatant to obtain walnut protein extract B;

[0101] (2) Mix walnut protein extract B with 50 mM phosphate buffer (containing 1.0 M KCl, pH=8.0) at a ratio of 1:1.5 (v / v), stir well, centrifuge, let stand for 15 min, collect the middle layer and the lower aqueous phase, mix them, and obtain walnut protein extract B.

[0102] (3) Adjust the pH of walnut protein extract B to 4.5, let it stand for 1.5 h, centrifuge, collect the supernatant and precipitate, mix them to obtain walnut protein isolate B; add anhydrous ethanol as solvent to 0.3 mM NaCl to make the final concentration 0.02 mM to obtain solution B; mix walnut protein isolate B and solution B at a ratio of 1 g: 25 mL (w / v), stir evenly, let it stand for 1.5 h, centrifuge, collect the precipitate to obtain walnut protein isolate B;

[0103] (4) Add 11 times the mass of deionized water to walnut protein isolate B, stir well, and then separate it through an ultrafiltration membrane with a membrane flux of 10000 Da. Collect the retentate, freeze dry it, and obtain walnut protein isolate B.

[0104] The basic components of walnut protein isolate B are shown in Table 1.

[0105] The amino acid composition of walnut protein isolate B is shown in Table 2.

[0106] The phenotypic properties of walnut protein isolate B are shown below. Figure 1 .

[0107] The solubility of walnut protein isolate B is shown in the figure. Figure 2 .

[0108] For the foaming properties / stability of walnut protein isolate B, see [link to relevant documentation]. Figure 3 .

[0109] The emulsifying properties of walnut protein isolate B are shown in [link to documentation]. Figure 4 .

[0110] The foaming properties of walnut protein isolate B are shown in [link to relevant documentation]. Figure 5 .

[0111] The digestibility of walnut protein isolate B in an elderly in vitro digestion model is shown in the figure. Figure 6 .

[0112] The AChE inhibition rate of walnut protein isolate B in an elderly in vitro digestion model is shown in the figure. Figure 7 .

[0113] The DPPH clearance rate of walnut protein isolate B in an elderly in vitro digestive model is shown in the figure. Figure 8 .

[0114] The ABTS clearance rate of walnut protein isolate B in an elderly in vitro digestion model is shown in the figure. Figure 9.

[0115] Example 3

[0116] A method for preparing walnut protein isolate includes the following steps:

[0117] (1) Dissolve sodium di(2-ethylhexyl)succinate sulfonate in n-hexane to prepare a solution A with a concentration of 0.18 M. Then mix 50 mM phosphate buffer (containing 0.1 MKCl, pH=7.7) with solution A at a ratio of 1:22 (v / v) and stir at 50 r / min for 30 min to form reverse micelles; then mix defatted walnut powder with the reverse micelles at a ratio of 1 g:20 mL (w / v), stir evenly, centrifuge, and collect the supernatant to obtain walnut protein extract C;

[0118] (2) Mix walnut protein extract C with 50 mM phosphate buffer (containing 1.0 M KCl, pH=8.2) at a ratio of 1:2 (v / v), stir well, centrifuge, let stand for 20 min, collect the middle layer and the lower aqueous phase, mix them, and obtain walnut protein extract C.

[0119] (3) Adjust the pH of walnut protein extract C to 4.5, let it stand for 2 h, centrifuge, collect the supernatant and precipitate, mix them to obtain walnut protein isolate C; add anhydrous ethanol as solvent to 0.5 mM NaCl to make the final concentration 0.03 mM to prepare solution B; mix walnut protein isolate C and solution B at a ratio of 1 g: 30 mL (w / v), stir evenly, let it stand for 2 h, centrifuge, collect the precipitate to obtain walnut protein isolate C;

[0120] (4) Add 12 times the mass of deionized water to walnut protein isolate C, stir well, and then separate it through an ultrafiltration membrane with a membrane flux of 10000 Da. Collect the retentate, freeze dry it, and obtain walnut protein isolate C.

[0121] The basic components of walnut protein isolate C are shown in Table 1.

[0122] The amino acid composition of walnut protein isolate C is shown in Table 2.

[0123] The phenotypic properties of walnut protein isolate C are shown in [reference needed]. Figure 1 .

[0124] The solubility of walnut protein isolate C is shown in the figure. Figure 2 .

[0125] For the foaming properties / stability of walnut protein isolate C, see [link to relevant documentation]. Figure 3 .

[0126] The emulsifying properties of walnut protein isolate C are shown in [link to documentation]. Figure 4 .

[0127] The foaming properties of walnut protein isolate C are shown in [reference needed]. Figure 5 .

[0128] The digestibility of walnut protein isolate C in an elderly in vitro digestion model is shown in the figure. Figure 6 .

[0129] The AChE inhibition rate of walnut protein isolate C in an elderly in vitro digestion model is shown in the figure. Figure 7 .

[0130] The DPPH clearance rate of walnut protein isolate C in an elderly in vitro digestive model is shown in the figure. Figure 8 .

[0131] The ABTS clearance rate of walnut protein isolate C in an elderly in vitro digestion model is shown in the figure. Figure 9 .

[0132] Comparative Example 1

[0133] A method for preparing walnut protein isolate, the comparative example differing from the previous one in that it does not use an acid precipitation and alcohol washing process to enrich and purify the walnut protein isolate, specifically including the following steps:

[0134] (1) Dissolve sodium di(2-ethylhexyl)succinate sulfonate in n-hexane to prepare a solution A with a concentration of 0.18 M. Then mix 50 mM phosphate buffer (containing 0.05 M KCl, pH=7.5) with solution A at a ratio of 1:18 (v / v) and stir at 30 r / min for 30 min to form reverse micelles; then mix defatted walnut powder with the reverse micelles at a ratio of 1 g:15 mL (w / v), stir evenly, centrifuge, and collect the supernatant to obtain walnut protein extract 1;

[0135] (2) Mix walnut protein extract 1 with an equal volume of 50 mM phosphate buffer (containing 1.0 M KCl, pH=8.0), stir well, centrifuge, let stand for 10 min, collect the middle layer and the lower aqueous phase, mix them, and obtain walnut protein extract 1.

[0136] (3) Add 10 times the mass of deionized water to walnut protein extract 1, stir evenly, and then separate it through an ultrafiltration membrane with a membrane flux of 10000 Da. Collect the retentate, freeze dry it, and obtain walnut protein isolate 1.

[0137] The basic components of walnut protein isolate 1 are shown in Table 1.

[0138] The amino acid composition of walnut protein isolate 1 is shown in Table 2.

[0139] The phenotypic characteristics of walnut protein isolate 1 are shown in [reference needed]. Figure 1 .

[0140] The solubility of walnut protein isolate 1 is shown in the figure. Figure 2 .

[0141] For the foaming properties / stability of walnut protein isolate 1, see [link to relevant documentation]. Figure 3 .

[0142] The emulsifying properties of walnut protein isolate 1 are shown in [link to documentation]. Figure 4 .

[0143] The foaming properties of walnut protein isolate 1 are shown in [link to documentation]. Figure 5 .

[0144] The digestibility of walnut protein isolate 1 in an elderly in vitro digestion model is shown in [reference needed]. Figure 6 .

[0145] The AChE inhibition rate of walnut protein isolate 1 in an elderly in vitro digestion model is shown in the figure. Figure 7 .

[0146] The DPPH clearance rate of walnut protein isolate 1 in an elderly in vitro digestive model is shown in the figure. Figure 8 .

[0147] The ABTS clearance rate of walnut protein isolate 1 in an elderly in vitro digestion model is shown in the figure. Figure 9 .

[0148] Comparative Example 2

[0149] A method for preparing walnut protein isolate, the comparative example differs from the embodiment in that it utilizes a traditional alkali-dissolving and acid-precipitating process to extract the walnut protein isolate, specifically including the following steps:

[0150] (1) After mixing defatted walnut powder with deionized water at a ratio of 1g:20mL (w / v), adjust the pH to 9, stir (150 r / min, 45℃, 2h), centrifuge, and collect the supernatant to obtain walnut protein extract 2.

[0151] (2) Adjust the pH of walnut protein extract 2 to 4.5, let it stand for 1 h, centrifuge, and collect the precipitate to obtain walnut protein isolate 2.

[0152] (3) Add 10 times the mass of deionized water to walnut protein isolate 2, stir evenly, and then separate through an ultrafiltration membrane with a membrane flux of 10000 Da. Collect the retentate, freeze dry, and obtain walnut protein isolate 2.

[0153] The basic components of walnut protein isolate 2 are shown in Table 1.

[0154] The amino acid composition of walnut protein isolate 2 is shown in Table 2.

[0155] The phenotypic characteristics of walnut protein isolate 2 are shown in [link to relevant documentation]. Figure 1 .

[0156] The solubility of walnut protein isolate 2 is shown in the figure. Figure 2 .

[0157] For the foaming properties / stability of walnut protein isolate 2, see [link to relevant documentation]. Figure 3 .

[0158] The emulsifying properties of walnut protein isolate 2 are shown in [link to documentation]. Figure 4 .

[0159] The foaming properties of walnut protein isolate 2 are shown in [link to documentation]. Figure 5 .

[0160] The digestibility of walnut protein isolate 2 in an elderly in vitro digestion model is shown in [reference needed]. Figure 6 .

[0161] The AChE inhibition rate of walnut protein isolate 2 in an elderly in vitro digestion model is shown in the figure. Figure 7 .

[0162] The DPPH clearance rate of walnut protein isolate 2 in an elderly in vitro digestive model is shown in [reference needed]. Figure 8 .

[0163] The ABTS clearance rate of walnut protein isolate 2 in an elderly in vitro digestion model is shown in the figure. Figure 9 .

[0164] Comparative Example 3

[0165] A method for preparing walnut protein isolate, the difference between this comparative example and the previous one being that the walnut protein isolate is prepared using a traditional alkaline dissolution dialysis process, specifically including the following steps:

[0166] (1) After mixing defatted walnut powder with deionized water at a ratio of 1g:20mL (w / v), adjust the pH to 9, stir (150 r / min, 45℃, 2h), centrifuge, and collect the supernatant to obtain walnut protein extract 3.

[0167] (2) Add 10 times the volume of deionized water to the walnut protein extract 3, stir well, and then separate it through an ultrafiltration membrane with a membrane flux of 10000 Da. Collect the retentate, freeze dry it, and obtain walnut protein isolate 3.

[0168] The basic components of walnut protein isolate 3 are shown in Table 1.

[0169] The amino acid composition of walnut protein isolate 3 is shown in Table 2.

[0170] The phenotypic characteristics of walnut protein isolate 3 are shown in [link to relevant documentation]. Figure 1 .

[0171] The solubility of walnut protein isolate 3 is shown in [reference needed]. Figure 2 .

[0172] For the foaming properties / stability of walnut protein isolate 3, see [link to relevant documentation]. Figure 3 .

[0173] The emulsifying properties of walnut protein isolate 3 are shown in [link to documentation]. Figure 4 .

[0174] The foaming properties of walnut protein isolate 3 are shown in [link to documentation]. Figure 5 .

[0175] The digestibility of walnut protein isolate 3 in an elderly in vitro digestion model is shown in [reference needed]. Figure 6 .

[0176] The AChE inhibition rate of walnut protein isolate 3 in an elderly in vitro digestion model is shown in the figure. Figure 7 .

[0177] The DPPH clearance rate of walnut protein isolate 3 in an elderly in vitro digestive model is shown in [reference needed]. Figure 8 .

[0178] The ABTS clearance rate of walnut protein isolate 3 in an elderly in vitro digestion model is shown in the figure. Figure 9 .

[0179] Table 1. Basic composition of walnut protein isolate (unit: %)

[0180]

[0181] Different letters indicate significant differences between samples. p <0.05)

[0182] Table 2. Amino acid content of walnut protein isolate (unit: mg / g)

[0183]

[0184] Different letters indicate significant differences between samples (p<0.05).

[0185] Using traditional alkaline solutions to dissolve proteins in raw materials (such as Comparative Examples 2 and 3) often results in the loss of some amino acids in walnut protein isolate, leading to a reduction in certain functional activities (reduced oral AChE inhibitory activity and antioxidant activity). In addition, it also results in a darker color of walnut protein isolate and poorer processing performance (low solubility, poor emulsification, and poor foaming properties), thus limiting the development of walnut protein isolate products.

[0186] This invention utilizes the reverse micelle method to extract walnut protein, and then uses acid precipitation and alcohol washing processes to enrich and purify the extracted walnut protein, in order to obtain a walnut protein isolate with significant properties for improving memory impairment in the elderly without damaging the processing characteristics of walnut protein.

[0187] As shown in Table 1, the protein purity of walnut protein isolates A to C obtained in the three embodiments of this invention is approximately 90%, comparable to that of walnut protein isolates 2 and 3 obtained using the traditional alkali-soluble acid precipitation and alkali-soluble dialysis methods. However, it is significantly higher than that of walnut protein isolate 1 obtained using the reverse micelle method reported in the literature. This is because the acid precipitation and alcohol washing processes in the three embodiments removed some impurities, resulting in enrichment and purification. The results demonstrate that the walnut protein isolate prepared by the method of this invention has the advantages of high purity and low impurity content.

[0188] The color of proteins and their products is a significant factor influencing consumer purchasing decisions; consumers tend to prefer protein products that are whiter and brighter. For example... Figure 1 The images of the protein powder and their L* (brightness), a* (red), and b* (yellow) values ​​are shown. Compared to the comparative examples walnut protein isolates 1-3, the walnut protein isolates A-C of the three examples are whiter and brighter. This is mainly because the extraction environment in the three examples is milder (compared to comparative examples 2 and 3), thereby reducing the oxidation reaction of polyphenols. In addition, the acid precipitation and alcohol washing processes also removed some polyphenols and reduced the subsequent ultrafiltration purification time, resulting in a weaker degree of polyphenol oxidation in the final walnut protein, thus obtaining a walnut protein that is whiter and brighter than that of comparative example 1. It can be seen that the walnut protein isolate obtained by the present invention exhibits better apparent properties.

[0189] Solubility is an important indicator for assessing the processing properties of proteins. Figure 2 It can be seen that the solubility trends of walnut protein isolates A-C in the examples and walnut protein isolates 1-3 in the comparative examples in water with pH (2.0-10.0) are roughly similar (pH-dependent U-shaped graph). The solubility of walnut protein isolates A-C in the three examples is higher than that of walnut protein isolates 1-3 in the pH range of 2.0-10.0. This may be because different extraction methods result in different degrees of conformational unfolding and dispersion of walnut protein aggregates, thereby affecting the distribution of hydrophobic groups on the protein surface, ultimately leading to differences in walnut protein solubility. In addition, acid precipitation and alcohol washing processes can reduce the residue of AOT on the surface of walnut protein and prevent walnut protein from undergoing aggregation reactions in aqueous solution, which is also the main reason why the solubility of walnut protein obtained in the three examples is higher than that of walnut protein in comparative example 1. High water solubility of protein is crucial for industrial applications under weakly alkaline, acidic, and neutral pH conditions. Therefore, the walnut protein isolate product prepared by this invention has good application potential in various industrial applications, especially in acidic food systems such as nutritional and sports drinks and acidified sauces.

[0190] Depend on Figure 3It can be seen that, compared with the comparative example walnut protein isolate 1, the AOT residue content of the three examples of walnut protein isolates A to C was significantly lower (approximately 15%). This is attributed to the ability of ethanol-NaCl to separate walnut protein isolate and surfactant. Ethanol can separate AOT from the AOT-walnut protein isolate complex and promote AOT dissolution. In addition, the presence of sodium ions helps to enhance the stability of AOT charge, thus preventing the rebinding of AOT with walnut protein isolate.

[0191] The emulsifying properties of proteins are extremely important for many processed foods, such as salad dressings and cakes. The Emulsifying Activity Index (EAI) represents the interfacial area produced per unit mass of protein as a surfactant. The Emulsion Stability Index (ESI) represents the stability of an emulsion against aggregation over time. Figure 4 It can be seen that the EAI values ​​of the three examples of walnut protein isolate A-C and the three comparative examples of walnut protein isolate 1-3 are similar. However, the three examples of walnut protein isolate A-C exhibit the highest ESI, which may be attributed to their excellent water solubility, and excellent water solubility is a prerequisite for proteins to perform emulsifying functions. Therefore, the walnut protein obtained by this invention has excellent emulsifying properties and is suitable for preparing emulsified foods such as cakes and ice cream.

[0192] Depend on Figure 5 It is evident that the walnut protein isolates A through C in the three examples exhibit excellent foaming ability, especially foaming stability. This may be because the walnut protein isolates in these examples have high solubility, causing the protein to rapidly unfold and fold around the gas or air, generating more bubbles. The walnut protein isolates A through C prepared in this invention exhibit excellent foaming ability and stability, and can be used in the processing of foamed foods such as cream, cakes, and foamed beverages, showing broad application prospects.

[0193] Table 2 shows the amino acid content results of the walnut protein isolates from the examples and comparative examples. Seventeen amino acids were detected in walnut protein isolates A–C from the examples and walnut protein isolates 1–3 from the comparative examples, including eight essential amino acids and nine non-essential amino acids. Except for tryptophan (not detected) and lysine (whose content was below the recommended level), the essential amino acid content of walnut protein isolates A–C and walnut protein isolates 1–3 all exceeded the recommended levels of the FAO / WHO / UN University (2007). Arginine is considered a key amino acid in neuroprotective peptides and can regulate AChE activity through specific interaction with the peripheral anion binding site of acetylcholinesterase (ACh). As shown in Table 2, the walnut protein isolates obtained in the three examples all had high arginine content (157.33±0.14~159.10±0.21 mg / g). This is because strong alkaline treatment (comparative examples 2 and 3) causes racemization of arginine in walnut protein, while reverse micelle extraction (examples and comparative example 1) is gentler and can effectively protect arginine during protein extraction. The arginine content of the walnut protein obtained in the examples and comparative examples differed significantly, mainly because the enrichment and purification process of acid precipitation and alkali washing reduced the ultrafiltration removal time and the loss of arginine in the protein during the removal process. These results indicate that the walnut protein isolate obtained in this invention has superior potential for improving memory impairment.

[0194] Statistics show that the elderly are the main group experiencing memory impairment. Therefore, characterizing the memory-improving effects of the prepared walnut protein isolate after digestion in the elderly is of great significance. Figure 6 This study demonstrates the in vitro digestibility of walnut protein isolates A-C (Examples 1-C) and walnut protein isolates 1-3 (Comparative Examples 1-3) in a simulated elderly digestion model. Significance analysis revealed that the in vitro digestibility of walnut protein isolates A-C (Examples 1-C) was relatively high. However, the digestibility of walnut protein in Comparative Example 1 was lower, likely because residual AOT (acid-dependent oxidizing agent) affected the digestive efficiency of gastrointestinal enzymes. As is well known, the nutritional value of protein is closely related to its digestibility; therefore, it is evident that the walnut protein isolates from the three examples provide enhanced nutritional value after oral administration.

[0195] Figure 7 The results show the AChE inhibitory activity of walnut isolates from the examples and comparative examples after in vitro gastrointestinal digestion. Similar to the results of the in vitro digestion experiment, walnut isolates A to C from the three examples showed stronger AChE inhibitory activity, which may be due to the following two reasons: (1) The higher in vitro digestibility means that the walnut isolates from the three examples can be broken down into more small molecule peptides, thereby increasing the probability of the release of AChE inhibitory peptides; (2) Walnut isolates 1 to 3 from examples have a higher arginine content (Table 2), so there may be more peptides with AChE inhibitory activity among the released small molecule peptides.

[0196] In addition, by Figure 8 and Figure 9 It can be seen that the walnut protein isolates from the three examples exhibit strong antioxidant properties, especially ABTS scavenging ability. This may be due to the release of more antioxidant peptides during digestion. However, it is noteworthy that the walnut protein isolates A-C obtained from the three examples showed no significant difference in DPPH scavenging ability compared to the walnut protein of Comparative Example 3. This is mainly because the walnut protein of Comparative Example 3 exposed more restriction enzyme sites during extraction due to structural unfolding, resulting in the production of more DPPH scavenging peptides after gastrointestinal digestion. The results indicate that the walnut protein isolate prepared in this invention has the potential to improve memory impairment in the elderly after oral administration.

[0197] In summary, the walnut protein isolates 1-3 of the three embodiments exhibit the best apparent properties, highest solubility, foaming stability, emulsification stability, arginine content, in vitro digestibility in elderly models, AChE inhibition rate after digestion, and antioxidant capacity, with low surfactant residue. Therefore, the walnut protein isolate preparation method of the present invention is an ideal solution that can improve memory impairment in the elderly, has excellent processing characteristics, and is suitable for large-scale industrial production.

[0198] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing walnut protein isolate, characterized in that... Includes the following steps: (1) Dissolve sodium di(2-ethylhexyl)succinate sulfonate in n-hexane to prepare solution A. Then mix buffer containing 0.05-0.1 M KCl with solution A and stir to form reverse micelle solution. Then mix defatted walnut powder with reverse micelle solution, stir evenly, centrifuge, and collect the supernatant to obtain walnut protein extract 1. (2) Mix walnut protein extract 1 with a buffer solution containing 0.8-1.0 M KCl, stir well, centrifuge, let stand to separate the layers, collect the middle layer and the lower aqueous phase, mix them to obtain walnut protein extract 2. (3) Adjust the pH of walnut protein extract 2 to 4.5, let it stand, centrifuge, collect the supernatant and precipitate, mix them to obtain walnut protein isolate 3; add anhydrous ethanol as solvent to 0.1-0.5 mM NaCl to make the final concentration reach 0.01-0.03 mM to prepare solution B; mix walnut protein isolate 3 with solution B, stir evenly, let it stand, centrifuge, collect the precipitate to obtain walnut protein isolate 4; In step (3), the walnut protein isolate 3 is mixed with solution B at a ratio of 1:(20-30) (w / v); (4) Add deionized water to walnut protein isolate 4, stir well, and then separate it through an ultrafiltration membrane with a membrane flux of 10000 Da. Collect the retentate, freeze dry it, and obtain walnut protein isolate.

2. The preparation method according to claim 1, characterized in that: The volume ratio of the buffer solution to solution A in step (1) is 1:(18-22).

3. The preparation method according to claim 1, characterized in that: In step (1), the defatted walnut powder and the reverse micelle solution are mixed at a ratio of 1:(15-20) (w / v).

4. The preparation method according to claim 1, characterized in that: In step (2), the volume ratio of walnut protein extract 1 to buffer solution is 1:(1-2).

5. A walnut protein isolate, characterized in that... It is prepared by the method described in any one of claims 1 to 4.

6. The application of the walnut protein isolate according to claim 5 in the preparation of health food products that help improve memory.

7. The application of the walnut protein isolate according to claim 5 in the preparation of health food products that help improve memory in the elderly.

8. The application of the walnut protein isolate according to claim 5 in the preparation of acidic foods.

9. The application of the walnut protein isolate according to claim 5 in the preparation of emulsified foods and foamed foods.

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