Preparation method of walnut protein isolate

Abstract

The invention discloses a preparation method of walnut protein isolate, and the walnut protein isolate prepared by the method has the efficacy of improving dysmnesia of old people and can be used for preparing acidic food, emulsified food and foam food. According to the method disclosed by the invention, the high-quality walnut protein isolate is prepared by taking degreased walnut powder as a raw material and utilizing the principles of reverse micelle dissolution, isoelectric precipitation and high solubility of bis (2-ethylhexyl) sodium sulfosuccinate in ethanol. The walnut protein isolate prepared by the invention has the advantages of high apparent property, high protein content, strong solubility, strong emulsifying ability, strong foaming ability, high in-vitro old-age model digestibility and AChE inhibition ability and antioxidant ability of strong digestion products.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of functional food, and particularly relates to a preparation method of walnut protein isolate. BACKGROUND

[0002] With the aggravation of population aging, the number of people suffering from neurocognitive diseases such as memory impairment has increased significantly, among which the proportion of elderly patients is the highest. Memory impairment is defined as a pathological state of persistent functional decline in any one or more cognitive links such as recognition, storage, maintenance and reproduction, and is the core symptom of neurodegenerative diseases such as Alzheimer's disease, vascular dementia and Lewy body dementia. This disease causes the progressive loss of patients' ability to live daily, which brings double pressure to the family care system and social medical security. Therefore, building a precise intervention and prevention system has become an urgent task with great public health value and social and economic benefits.

[0003] Walnut meal is a by-product produced in the process of walnut oil extraction. It not only has a high protein content (more than 40%), but also has a unique amino acid composition characteristic (rich in arginine and balanced essential amino acid ratio), which is highly consistent with the demand for neurotransmitter synthesis, and is an excellent raw material source for developing memory function improving preparations. Therefore, it is of great significance to extract walnut protein isolate from walnut meal using modern processing technology for the development of functional foods with potential cognitive improvement function.

[0004] At present, the alkali-soluble acid precipitation process is widely used in the preparation of walnut protein isolate due to its simple operation and low cost. However, alkaline conditions may cause peptide chain breakage and promote covalent modification reactions, including the aggregation effect of amino acid residues, beta-elimination reaction and racemization phenomenon. And excessive alkali treatment will also cause abnormal unfolding of protein conformation and changes in specific amino acid residues (such as arginine, lysine and serine). It is worth noting that some studies have shown that arginine derivatives have good protective effect on nerve cells, and their functional integrity directly affects the cognitive improvement effect. Therefore, the traditional extraction process may reduce the neuroprotective activity of walnut protein isolate.

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

[0006] 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 acidic food, emulsified food and foamed food. The method uses defatted walnut powder (walnut meal) as raw material, utilizes reverse micelle dissolution, isoelectric point precipitation and the principle of high solubility of sodium 2-ethylhexyl sulfosuccinate (AOT) in ethanol to prepare high-quality walnut protein isolate. Specifically, AOT, n-hexane and phosphate buffer are mixed at a certain proportion to prepare a reverse micelle solution, and defatted walnut powder is dissolved in the reverse micelle solution; the supernatant is mixed with an equal amount of phosphate buffer and stirred through centrifugal separation; the mixture forms a three-layer system after centrifugal separation and standing; the interface layer and the lower water phase are collected and uniformly mixed, and the pH of the mixture is adjusted to the isoelectric point of walnut protein to precipitate the protein; the precipitate and the upper suspension are collected and mixed after further centrifugal separation; ethanol-NaCl solvent is added to the mixture to separate the protein from AOT; after centrifugal separation, the precipitate is mixed with a small amount of water to be redissolved into a protein solution, and further ultrafiltration is performed to remove small molecular impurities; the protein solution is freeze-dried to obtain the walnut protein isolate.

[0007] The application achieves the purpose by the following technical scheme. The walnut protein isolate preparation method comprises the following steps. (1) Sodium 2-ethylhexyl sulfosuccinate is dissolved in n-hexane to prepare solution A, then a buffer solution containing 0.05-0.1 M KCl is mixed with solution A to form a reverse micelle solution through stirring; defatted walnut powder is mixed with the reverse micelle solution, stirred uniformly, centrifuged, and the supernatant is collected to obtain walnut protein extract 1; The concentration of solution A in step (1) is preferably 0.18 M; The volume ratio of the buffer solution to solution A in step (1) is preferably 1:(18-22); The defatted walnut powder is mixed with the reverse micelle solution in step (1) at a ratio of 1:(15-20) (w / v); The buffer solution in step (1) is a phosphate buffer or a Tris-HCl buffer, and the pH value is 7.3-7.7; The defatted walnut powder in step (1) is a powdery material with an oil content of less than 5% obtained by crushing walnut kernels as raw material through pressing and / or solvent leaching treatment, and is also called walnut meal.

[0008] Centrifugation in step (1) can be performed by using a tubular centrifuge or a disc centrifuge to ensure that no obvious precipitate appears in the supernatant under a centrifugal force of 5000xg for 5 min; (2) mixing the walnut protein extract 1 with a buffer solution containing 0.8-1.0 M KCl, stirring uniformly, and then centrifuging and separating into layers, collecting the middle layer and the lower water phase, mixing, to obtain a walnut protein extract 2; The volume ratio of the walnut protein extract 1 to the buffer solution in step (2) is preferably 1:(1-2). The buffer solution in step (2) is a phosphate buffer solution or a Tris-HCl buffer solution, and the pH value is 7.8-8.2. The stirring conditions in steps (1) and (2) are as follows: temperature 35-45 ℃, frequency 180-200 rpm, and time 30 min-60 min.

[0009] (3) adjusting the pH value of the walnut protein extract 2 to 4.5, standing, centrifuging, collecting the upper layer and the precipitate, mixing, to obtain a walnut protein isolate 3; adding 0.1-0.5 mM NaCl in anhydrous ethanol as a solvent, so that the final concentration reaches 0.01-0.03 mM, to prepare a solution B; mixing the walnut protein isolate 3 with the solution B, stirring uniformly, standing, centrifuging, and collecting the precipitate to obtain a walnut protein isolate 4. The walnut protein isolate 3 is preferably mixed with the solution B in a ratio of 1:(20-30) (w / v) in step (3). (4) adding deionized water in the walnut protein isolate 4, stirring uniformly, and then separating through an ultrafiltration membrane with a membrane flux of 10000 Da, collecting the retentate, and freeze-drying, to obtain a walnut protein isolate.

[0010] The walnut protein isolate prepared by the above method has a high arginine content, strong antioxidant activity and AChE inhibitory activity, and a high in vitro digestion rate under a simulated old person digestion model, and can be used for preparing medicines and health foods for assisting in improving memory. Further, the walnut protein isolate prepared by the above method can be used for preparing medicines and health foods for assisting in improving memory of old people.

[0011] The walnut protein isolate prepared by the above method has a high water solubility under acidic conditions, and can be used for preparing acidic foods, including nutritional and sports beverages, acidified sauces and the like. The walnut protein isolate prepared by the above method has excellent emulsifying properties, and excellent foaming ability and stability, and can be used for preparing emulsified foods and foamed foods.

[0012] The present application has the following advantages and effects relative to the prior art: 1、The walnut protein isolate is extracted and enriched by adopting the reverse micelle method combined with acid precipitation and alcohol washing process, compared with the traditional alkali extraction, the amino acid loss can be reduced and the processing characteristics can be improved, the method can be used for industrial production and can serve experimental theory research, and has outstanding innovation.

[0013] 2、The acid precipitation and alcohol washing process of adding non-toxic and harmless hydrochloric acid solution and ethanol-NaCl solution is introduced on the basis of the existing reverse micelle method for extracting protein, the residual surfactant in the walnut protein isolate can be reduced, and the process is safe, efficient, green, environmentally friendly and sustainable.

[0014] 3、The walnut protein isolate prepared has the advantages of high-quality apparent properties, high protein content, strong solubility, strong emulsifying capacity, strong foaming capacity, high in-vitro elderly model digestion rate, and strong AChE inhibition capacity and antioxidant capacity of digestion products. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is the apparent property of walnut protein isolate; Figure 2 is the solubility of walnut protein isolate; Figure 3 is the AOT residual amount of walnut protein isolate; Figure 4 is the emulsifying property of walnut protein isolate; Figure 5 is the foaming property of walnut protein isolate; Figure 6 is the digestion rate of walnut protein isolate in an in-vitro elderly digestion model; Figure 7 is the AChE inhibition rate of walnut protein isolate in an in-vitro elderly digestion model; Figure 8 is the DPPH clearance rate of walnut protein isolate in an in-vitro elderly digestion model; Figure 9 is the ABTS clearance rate of walnut protein isolate in an in-vitro elderly digestion model; 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 represent significant differences (p<0.05) between samples. DETAILED DESCRIPTION

[0016] The application will be further described in detail below in combination with examples and drawings, but the embodiments of the application are not limited thereto.

[0017] The experimental methods of the test indexes involved in the embodiments of the application are as follows: 1、Walnut protein isolate component test (1) Protein purity determination: Take 2 g of walnut protein isolate, use the Kjeldahl method (refer to GB 5009.5-2016) to determine the protein content, and calculate the protein purity of walnut protein isolate according to equation (1): Protein purity (%) = Protein content of walnut protein isolate / Mass of walnut protein isolate x 100 (1) (2) Total sugar content determination: Prepare a glucose standard stock solution, use the phenol-sulfuric acid method to draw a standard curve. Take an appropriate amount of walnut protein isolate, according to the operation method of the standard curve, perform three parallel experiments, then measure the absorbance value at 595 nm, and calculate the total sugar content of the walnut protein isolate.

[0018] (3) Ash content is determined according to GB 5009.4-2016, moisture content is determined according to GB 5009.3-2016, and fat content is determined according to GB 5009.6-2016.

[0019] 2. Determination of color The color of walnut protein isolate was measured using a portable precision color difference meter (model WSC-2B, Shanghai Yiqian Physical Optics Instrument Co., Ltd.).

[0020] 3. Determination of AOT Prepare 0.007% methylene blue with 1% Na2SO4 aqueous solution, then mix 1 mL methylene blue solution with 5 mL chloroform. Methylene blue forms a salt with AOT, which dissolves in chloroform to form a colored layer. Remove the upper aqueous phase containing excess dye. Transfer the chloroform phase into a cuvette and measure its absorbance on a UV spectrophotometer at 650 nm. Calculate the AOT content in the protein using the AOT standard curve.

[0021] 4. Method for determining the solubility of walnut protein isolate Prepare a suspension by mixing walnut protein isolate and ultrapure water at a ratio of 1:20 (w / v). Adjust the suspension to pH 2.0-12.0 using 0.1 M HCl or 0.1 M NaOH. After mixing the suspension, centrifuge it (4000 g, 30 min, 25°C). Measure the protein content of the supernatant by the Kjeldahl method, and calculate the solubility according to equation (2): Solubility (%) = Protein content in supernatant / Protein content in walnut protein isolate sample x 100 (2) 5. Determination of emulsifying capacity Emulsification properties including emulsion activity index (EAI) and emulsion stability index (ESI) were determined by turbidimetry. At room temperature, 30 mL of protein solution (0.2%, w / v) was mixed with 10 mL of soybean oil using a homogenizer (Model T25, IKA Werke GmbH, Germany) at 10000 x g for 1 minute. At 0 and 10 minutes after homogenization, emulsion (40 μL) was removed from the bottom of the container and dispersed into 5 mL of 0.1% SDS (w / v). The absorbance at 500 nm wavelength was measured by spectrophotometry. EAI and ESI were calculated according to equations (3) and (4), respectively: EAI (m 2 / g) = 2 × 2.303 / (c × φ (1-θ) × 10 4 ) × A0× dilution factor (3) ESI (%) = A 10 / A0× 100 (4) where A0and A 10 are the absorbance at 0 and 10 minutes after 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 volume fraction of oil (0.20).

[0022] 6. Measurement of foaming capacity / stability Walnut protein isolate and ultra-pure water were mixed at a ratio of 1:100 (w / v) to prepare a suspension (V1). After homogenization at 11520 x g for 2 minutes using a homogenizer, 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. Foaming capacity and foam stability were calculated using equations (5) and (6): Foaming capacity (%) = (V2+V3-V1) / V1 x 100 (5) Foam stability (%) = V4 / V2 x 100 (6) 7. Measurement of hydrolyzed amino acids After hydrolysis of the sample at 110 °C for 24 hours using 6 M hydrochloric acid, an automatic amino acid analyzer (Model L-8900, Hitachi Instruments Service, Japan) was used for measurement.

[0023] 8. Establishment of an in vitro model of aging digestion and determination of digestibility 3 g of walnut protein isolate was digested in 3 mL of simulated saliva at a rotation speed of 7 rpm for 2 min. Then 6 mL of simulated gastric juice (containing 1500 U / mL pepsin) was added, and the pH value was adjusted to 4.5 with 5 mol / L HCl solution, the rotation speed was 7 rpm, and the digestion time was 180 min. Finally, 12 mL of simulated intestinal juice (containing 59.4 mg / mL bovine bile salt, 46 U / mL trypsin, and 23 U / mL pancreatic lipase) was added, the pH value was adjusted to 6.6 with 1 mol / L NaOH, the rotation speed was 7 rpm, and the digestion time was 180 min. After the digestion was completed, all samples were treated at 90℃ for 10 min to inactivate the enzyme. After thawing, all digests were centrifuged at 4℃, 6000 r / min for 15 min, and the protein content in the supernatant was further determined using a BCA protein kit, and the digestion rate was calculated according to equation (7).

[0024] Digestion rate (%) = protein content in supernatant / protein content of walnut protein isolate used for digestion × 100 (7) 9. Determination of AChE inhibition rate 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 a 96-well plate, mixed well, and incubated at 37℃ for 15 min. After incubation, 30 μL of AChE (0.055 U / mL) was added to start the determination. The determination was carried out in a microplate reader. The determination wavelength was 412 nm, and the determination time was 15 min. The calculation formula of the AChE inhibition activity of the sample is shown in equation (8): AChE inhibition activity (%) = [1- (A 样品 -A 样品空白 / A 对照 -A 对照空白 )] × 100 (8) Wherein, A 样品 is the absorbance of the sample group, A 样品空白 is the absorbance of the sample group without enzyme, A 对照 is the absorbance of the sample group with enzyme, and A 对照空白 is the absorbance of the sample group without enzyme.

[0025] 10. Determination of DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging ability 2 mL of sample solution was mixed with 2.0 ml of DPPH solution (0.2 mM). Then the mixture was vortexed and kept in the dark for 30 minutes. The absorbance at 517 nm was measured by spectrophotometry. The DPPH free radical scavenging activity of each protein sample was calculated according to equation (9): DPPH radical scavenging activity (%) = [1 - (A 样品 -A 空白 )] / A 对照 x 100 (9) wherein A 样品 , A 对照 and A 空白 are the absorbance values of the protein sample, control and blank, respectively.

[0026] 11. Assay for ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) scavenging capacity The absorbance of ABTS (7 mM ABTS and 2.45 mM potassium persulfate) at 734 nm was adjusted to 0.70 ± 0.02 with 75 mM phosphate buffer (pH 7.4). Then 50 μL of sample solution was added to 150 μL of diluted ABTS solution to determine the ABTS·removal activity. The ABTS radical scavenging activity of each protein sample was calculated using equation (10): ABTS radical scavenging activity (%) = [1 - (A 样品 -A 空白 )] / A 空白 x 100 (10) wherein A 样品 is the absorbance of the sample and A 空白 is the absorbance of the blank.

[0027] Example 1 A method for preparing walnut protein isolate, comprising the following steps: (1) Dissolve sodium bis(2-ethylhexyl) sulfosuccinate 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 = 7.3) 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 uniformly, centrifuge, and collect the supernatant to obtain walnut protein extract A; (2) Mix walnut protein extract A with 50 mM phosphate buffer (containing 1.0 M KCl, pH = 7.8) in equal volumes, stir uniformly, centrifuge, and let stand for 10 min. Collect the intermediate layer and lower aqueous phase, mix, and obtain walnut protein extract A; (3) The pH of the walnut protein extract A was adjusted to 4.5, and after standing for 1 h, the supernatant and precipitate were collected by centrifugation, mixed, and walnut protein isolate A was obtained; 0.1 mM NaCl was added to anhydrous ethanol as a solvent to make a solution B, with a final concentration of 0.01 mM; walnut protein isolate A was mixed with solution B at a ratio of 1 g:20 mL (w / v), stirred uniformly, and after standing for 1 h, the precipitate was collected by centrifugation to obtain walnut protein isolate A; (4) Deionized water was added to walnut protein isolate A at 10 times the mass of walnut protein isolate A, stirred uniformly, and then separated by an ultrafiltration membrane with a membrane flux of 10,000 Da, and the retentate was collected, freeze-dried, and walnut protein isolate A was obtained.

[0028] The basic components of walnut protein isolate A are shown in Table 1.

[0029] The amino acid composition of walnut protein isolate A is shown in Table 2.

[0030] The apparent properties of walnut protein isolate A are shown in Figure 1 .

[0031] The solubility of walnut protein isolate A is shown in Figure 2 .

[0032] The foaming properties / stability of walnut protein isolate A are shown in Figure 3 .

[0033] The emulsifying properties of walnut protein isolate A are shown in Figure 4 .

[0034] The foaming properties of walnut protein isolate A are shown in Figure 5 .

[0035] The digestibility of walnut protein isolate A in an in vitro digestion model of the elderly is shown in Figure 6 .

[0036] The AChE inhibition rate of walnut protein isolate A in an in vitro digestion model of the elderly is shown in Figure 7 .

[0037] The DPPH scavenging rate of walnut protein isolate A in an in vitro digestion model of the elderly is shown in Figure 8 .

[0038] The ABTS scavenging rate of walnut protein isolate A in an in vitro digestion model of the elderly is shown in Figure 9 .

[0039] Example 2 A method for preparing a walnut protein isolate includes the following steps: (1) Dissolve sodium bis(2-ethylhexyl) sulfosuccinate in n-hexane to prepare a solution A with a concentration of 0.18 M. Then mix 50 mM phosphate buffer (containing 0.075 M KCl, 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 uniformly, centrifuge, and collect the supernatant to obtain walnut protein extract B; (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 uniformly, centrifuge, and stand for 15 min, then mix the intermediate layer and the lower aqueous phase to obtain walnut protein extract B; (3) Adjust the pH of walnut protein extract B to 4.5, stand for 1.5 h, centrifuge, collect the upper layer and the precipitate, mix, and obtain walnut protein isolate B; add 0.3 mM NaCl to anhydrous ethanol to prepare solution B with a final concentration of 0.02 mM; mix walnut protein isolate B with solution B at a ratio of 1 g:25 mL (w / v), stir uniformly, stand for 1.5 h, centrifuge, and collect the precipitate to obtain walnut protein isolate B; (4) Add 11 times the mass of deionized water to walnut protein isolate B, stir uniformly, then separate by an ultrafiltration membrane with a membrane flux of 10,000 Da, collect the retentate, and freeze-dry to obtain walnut protein isolate B.

[0040] The basic components of walnut protein isolate B are shown in Table 1.

[0041] The amino acid composition of walnut protein isolate B is shown in Table 2.

[0042] The apparent properties of walnut protein isolate B are shown in Figure 1 .

[0043] The solubility of walnut protein isolate B is shown in Figure 2 .

[0044] The foaming properties / stability of walnut protein isolate B are shown in Figure 3 .

[0045] The emulsifying properties of walnut protein isolate B are shown in Figure 4 .

[0046] The foaming properties of walnut protein isolate B are shown in Figure 5 .

[0047] The digestibility of walnut protein isolate B in an in vitro digestion model for the elderly is shown in Figure 6 .

[0048] The AChE inhibition rate of walnut protein isolate B in the elderly in vitro digestion model is shown in Table 1. Figure 7 .

[0049] The DPPH scavenging rate of walnut protein isolate B in the elderly in vitro digestion model is shown in Table 2. Figure 8 .

[0050] The ABTS scavenging rate of walnut protein isolate B in the elderly in vitro digestion model is shown in Table 3. Figure 9 .

[0051] Example 3 A method for preparing walnut protein isolate, comprising the following steps: (1) Dissolve sodium bis(2-ethylhexyl) sulfosuccinate in n-hexane to prepare a solution A with a concentration of 0.18 M. Then mix 50 mM phosphate buffer (containing 0.1 M KCl, pH = 7.7) with solution A at a ratio of 1:22 (v / v), 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 uniformly, centrifuge, and collect the supernatant to obtain walnut protein extract C; (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 uniformly, centrifuge, and stand for 20 min, then mix the intermediate layer and the lower aqueous phase to obtain walnut protein extract C; (3) Adjust the pH of walnut protein extract C to 4.5, stand for 2 h, centrifuge, collect the upper layer and the precipitate, mix, and obtain walnut protein isolate C; add 0.5 mM NaCl to anhydrous ethanol as a solvent to make solution B with a final concentration of 0.03 mM; mix walnut protein isolate C with solution B at a ratio of 1 g:30 mL (w / v), stir uniformly, stand for 2 h, centrifuge, and collect the precipitate to obtain walnut protein isolate C; (4) Add 12 times the mass of deionized water to walnut protein isolate C, stir uniformly, then separate through an ultrafiltration membrane with a membrane flux of 10,000 Da, collect the retentate, freeze-dry, and obtain walnut protein C.

[0052] The basic components of walnut protein C are shown in Table 1.

[0053] The amino acid composition of walnut protein C is shown in Table 2.

[0054] The apparent properties of walnut protein C are shown in Table 3. Figure 1 .

[0055] The solubility of walnut protein C is shown in Table 4. Figure 2 .

[0056] Foaming properties / stability of walnut protein isolate C Figure 3 .

[0057] Emulsifying properties of walnut protein isolate C Figure 4 .

[0058] Foaming properties / stability of walnut protein isolate C Figure 5 .

[0059] Digestibility of walnut protein isolate C in an in vitro digestion model for the elderly Figure 6 .

[0060] AChE inhibitory activity of walnut protein isolate C in an in vitro digestion model for the elderly Figure 7 .

[0061] DPPH scavenging activity of walnut protein isolate C in an in vitro digestion model for the elderly Figure 8 .

[0062] ABTS scavenging activity of walnut protein isolate C in an in vitro digestion model for the elderly Figure 9 .

[0063] Comparative Example 1 A method for preparing walnut protein isolate, which differs from the examples in that the walnut protein isolate is not enriched and purified using an acid precipitation-alcohol washing process, and specifically comprises the following steps: (1) Dissolve sodium bis(2-ethylhexyl)sulfosuccinate 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 well, centrifuge, and collect the supernatant to obtain walnut protein extract 1; (2) Mix walnut protein extract 1 with 50 mM phosphate buffer (containing 1.0 M KCl, pH = 8.0) at equal volumes, stir well, centrifuge, and then let stand for 10 min. Collect the intermediate layer and the lower aqueous phase, mix, and obtain walnut protein extract 1; (3) Add 10 times the mass of deionized water to walnut protein extract 1, stir well, then separate by ultrafiltration membrane with a membrane flux of 10,000 Da, collect the retentate, and freeze-dry to obtain walnut protein isolate 1.

[0064] The basic components of walnut protein isolate 1 are shown in Table 1.

[0065] The amino acid composition of walnut protein isolate 1 is shown in Table 2.

[0066] The apparent properties of walnut protein isolate 1 are shown in Table 3. Figure 1 .

[0067] The solubility of walnut protein isolate 1 is shown in Table 4. Figure 2 .

[0068] The foaming properties / stability of walnut protein isolate 1 are shown in Table 5. Figure 3 .

[0069] The emulsifying properties of walnut protein isolate 1 are shown in Table 6. Figure 4 .

[0070] The foaming properties of walnut protein isolate 1 are shown in Table 7. Figure 5 .

[0071] The digestibility of walnut protein isolate 1 in the elderly in vitro digestion model is shown in Table 8. Figure 6 .

[0072] The AChE inhibition rate of walnut protein isolate 1 in the elderly in vitro digestion model is shown in Table 9. Figure 7 .

[0073] The DPPH scavenging rate of walnut protein isolate 1 in the elderly in vitro digestion model is shown in Table 10. Figure 8 .

[0074] The ABTS scavenging rate of walnut protein isolate 1 in the elderly in vitro digestion model is shown in Table 11. Figure 9 .

[0075] Comparative Example 2 A method for preparing walnut protein isolate, which is different from the examples in that walnut protein isolate is extracted by using a traditional alkali-solvent acid-precipitation process, specifically comprising the following steps: (1) Defatted walnut powder is mixed with deionized water at 1 g:20 mL (w / v), the pH is adjusted to 9, stirring (150 r / min, 45°C, 2h), centrifugation, and the supernatant is collected to obtain walnut protein extract 2.

[0076] (2) The pH of walnut protein extract 2 is adjusted to 4.5, and after standing for 1 h, centrifugation, and the precipitate is collected to obtain walnut protein isolate 2.

[0077] (3) 10 times the mass of deionized water is added to walnut protein isolate 2, stirred uniformly, and then separated by an ultrafiltration membrane with a membrane flux of 10000 Da, the retentate is collected, and freeze-dried to obtain walnut protein isolate 2.

[0078] The basic components of walnut protein isolate 2 are shown in Table 1.

[0079] The amino acid composition of walnut protein isolate 2 is shown in Table 2.

[0080] The apparent properties of walnut protein isolate 2 are shown in Table 3. Figure 1 .

[0081] Solubility of walnut protein isolate 2 is shown in Table 6. Figure 2 .

[0082] Foaming properties / stability of walnut protein isolate 2 is shown in Table 7. Figure 3 .

[0083] Emulsifying properties of walnut protein isolate 2 is shown in Table 8. Figure 4 .

[0084] Foaming properties of walnut protein isolate 2 is shown in Table 9. Figure 5 .

[0085] Digestibility of walnut protein isolate 2 in the elderly in vitro digestion model is shown in Table 10. Figure 6 .

[0086] AChE inhibitory rate of walnut protein isolate 2 in the elderly in vitro digestion model is shown in Table 11. Figure 7 .

[0087] DPPH scavenging rate of walnut protein isolate 2 in the elderly in vitro digestion model is shown in Table 12. Figure 8 .

[0088] ABTS scavenging rate of walnut protein isolate 2 in the elderly in vitro digestion model is shown in Table 13. Figure 9 .

[0089] Comparative Example 3 A method for preparing walnut protein isolate, which is different from the examples in that the walnut protein isolate is prepared by using a traditional alkali solubilization and dialysis process, specifically comprising the following steps: (1) The defatted walnut powder is mixed with deionized water at 1 g:20 mL (w / v), the pH is adjusted to 9, stirred (150 r / min, 45°C, 2h), centrifuged, and the supernatant is collected to obtain walnut protein extract 3.

[0090] (2) 10 times the volume of deionized water is added to the walnut protein extract 3, stirred uniformly, then separated by an ultrafiltration membrane with a membrane flux of 10000 Da, the retentate is collected, freeze-dried, and walnut protein isolate 3 is obtained.

[0091] The basic components of walnut protein isolate 3 are shown in Table 1.

[0092] The amino acid composition of walnut protein isolate 3 is shown in Table 2.

[0093] The apparent properties of walnut protein isolate 3 are shown in Table 5. Figure 1 .

[0094] Solubility of walnut protein isolate 3 is shown in Table 6. Figure 2 .

[0095] Foaming properties / stability of walnut protein isolate 3 is shown in Table 7.Figure 3 .

[0096] Emulsifying properties of walnut protein isolate 3 are shown in Table 4 Figure 4 .

[0097] Foaming properties of walnut protein isolate 3 are shown in Table 5 Figure 5 .

[0098] Digestibility of walnut protein isolate 3 in the elderly in-vitro digestion model is shown in Table 6 Figure 6 .

[0099] AChE inhibitory rate of walnut protein isolate 3 in the elderly in-vitro digestion model is shown in Table 7 Figure 7 .

[0100] DPPH scavenging rate of walnut protein isolate 3 in the elderly in-vitro digestion model is shown in Table 8 Figure 8 .

[0101] ABTS scavenging rate of walnut protein isolate 3 in the elderly in-vitro digestion model is shown in Table 9 Figure 9 .

[0102] Table 1 Basic composition of walnut protein isolate (unit: %)

[0103] Different letters indicate significant differences between samples (p<0.05) p <0.05) Table 2 Amino acid content of walnut protein isolate (unit: mg / g)

[0104] Different letters indicate significant differences between samples (p<0.05) Using traditional lye to dissolve the protein in the raw material (such as Comparative Examples 2 and 3) often causes loss of some amino acids in the walnut protein isolate, thereby reducing certain functional activities (reducing oral AChE inhibitory activity and antioxidant activity); in addition, it also causes the walnut protein isolate to have a darker color and poor processing performance (low solubility, poor emulsifying and foaming properties), thereby limiting the development of walnut protein isolate products.

[0105] The present application uses reverse micelle method to extract walnut protein, and uses acid precipitation and alcohol washing process to enrich and purify the extracted walnut protein, so as to obtain a walnut protein isolate with obvious memory impairment improvement for the elderly without destroying the processing properties of the walnut protein.

[0106] From the composition of walnut protein isolate in Table 1, it can be seen that the protein purity of the walnut protein isolates A-C obtained in the three embodiments of the present application is about 90%, which is comparable to the purity of the walnut protein isolates 2 and 3 prepared by the traditional alkali dissolution and acid precipitation and dialysis method, but is significantly higher than the walnut protein isolate 1 prepared by the reverse micelle method reported in the literature. This is because the acid precipitation and alcohol washing process in the three embodiments removes part of the impurities, so that the enrichment and purification are obtained. The results show that the walnut protein isolate prepared by the method of the present application has the advantages of high purity and less impurities.

[0107] The color of protein and its products is one of the important factors affecting the purchase of consumers, and consumers tend to purchase protein products with whiter and brighter color. Figure 1 The photos of the protein powders and their L* (brightness), a* (red) and b* (yellow) values are shown. Compared with the walnut protein isolates 1-3 of the comparative examples, the walnut protein isolates A-C of the three embodiments have whiter color and higher brightness, which is mainly because the extraction environment in the three embodiments is more mild (compared with the comparative examples 2 and 3), thereby reducing the oxidation reaction of polyphenols. In addition, the acid precipitation and alcohol washing process also removes part of the polyphenols and reduces the subsequent ultrafiltration time for removing impurities, resulting in a weaker oxidation degree of polyphenols in the final walnut protein, so that the walnut protein obtained has whiter color and higher brightness than the comparative example 1. It can be seen that the walnut protein obtained by the present application exhibits better apparent properties.

[0108] Solubility is an important indicator for evaluating the processing properties of protein. From the solubility of the walnut protein isolates A-C of the three embodiments and the walnut protein isolates 1-3 of the comparative examples in water at different pH values (2.0-10.0) shown in Table 2, it can be seen that the solubility of the walnut protein isolates A-C of the three embodiments and the walnut protein isolates 1-3 of the comparative examples in water changes with the pH value (2.0-10.0) in a similar trend (pH-dependent U-shaped graph). Figure 2 The solubility of the walnut protein isolates A-C of the three embodiments is higher than that of the walnut protein isolates 1-3 of the comparative examples in the pH range of 2.0-10.0. This may be because different extraction methods cause different degrees of unfolding and dispersion of walnut protein aggregates, thereby affecting the distribution of hydrophobic groups on the protein surface, and finally resulting in differences in solubility of walnut protein. In addition, the acid precipitation and alcohol washing process can reduce the residual of AOT on the surface of walnut protein, and prevent the aggregation reaction of walnut protein in aqueous solution, which is also the main reason why the solubility of the walnut protein obtained in the three embodiments is higher than that of the walnut protein of the comparative example 1. Under weakly alkaline, acidic and neutral pH conditions, high water solubility of protein is crucial for industrial applications. Therefore, the walnut protein isolate product prepared by the present application has good application potential in various industrial applications, especially in the application of acid food systems such as nutritional and sports beverages and acidified sauces.

[0109] From the solubility of the walnut protein isolates A-C of the three embodiments and the walnut protein isolates 1-3 of the comparative examples in water at different pH values (2.0-10.0) shown in Table 2, it can be seen that the solubility of the walnut protein isolates A-C of the three embodiments and the walnut protein isolates 1-3 of the comparative examples in water changes with the pH value (2.0-10.0) in a similar trend (pH-dependent U-shaped graph). Figure 3It can be seen that the AOT residual content of the three example walnut protein isolates A-C is significantly lower (about 15%) than that of the comparative walnut protein isolate 1, which is due 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 the dissolution of AOT. In addition, the presence of sodium ions helps to enhance the stability of the AOT charge, playing a role in preventing the recombination of AOT and walnut protein isolate.

[0110] 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 the emulsion to coalescence over time. The EAI and ESI were calculated from the following equations: Figure 4 It can be seen that the EAI values of the three example walnut protein isolates A-C and the three comparative walnut protein isolates 1-3 are similar. However, the three example walnut protein isolates A-C exhibit the highest ESI, which may be due to their excellent water solubility, which is a prerequisite for proteins to function as emulsifiers. It can be seen that the walnut protein obtained by the present application has excellent emulsifying properties and is suitable for the preparation of emulsified foods such as cakes and ice cream.

[0111] The EAI and ESI of the walnut protein isolates were calculated from the following equations: Figure 5 It can be seen that the three example walnut protein isolates A-C exhibit excellent foaming capacity, especially foaming stability. This may be because the solubility of the example walnut protein isolates is higher, resulting in rapid unfolding and folding of the protein around gas or air and the generation of more bubbles. The walnut protein isolates A-C prepared by the present application exhibit excellent foaming capacity and stability and can be used in the processing of foamed foods such as whipped cream, cakes, and foamed beverages, and have a wide application prospect.

[0112] Table 2 is the amino acid content results of the walnut protein isolates of the examples and the comparative examples. The example walnut protein isolates A-C and the comparative example walnut protein isolates 1-3 were detected for 17 amino acids, including 8 essential amino acids and 9 non-essential amino acids. Except for tryptophan (not detected) and lysine (the content of which was lower than the recommended level), the essential amino acid content of the example walnut protein isolates A-C and the comparative example walnut protein isolates 1-3 all exceeded the recommended level of the Food and Agriculture Organization of the United Nations / World Health Organization / United Nations University (2007). Arginine is considered to be a key amino acid of neuroprotective peptides, which can regulate AChE activity through specific interaction with peripheral anion binding sites of acetylcholinesterase (ACh). As can be seen from Table 2, the three example walnut protein isolates all have a relatively high arginine content (157.33±0.14-159.10±0.21 mg / g). This is because strong alkali treatment (comparative examples 2 and 3) can cause racemization of arginine in walnut protein, while the reverse micelle extraction method (examples and comparative example 1) is relatively mild and can effectively protect arginine during protein extraction. The arginine content of the walnut protein obtained in the examples and the comparative examples is significantly different, mainly because the enrichment and purification process of acid precipitation and alkali washing can reduce the ultrafiltration time for impurity removal and reduce the loss of arginine in the protein during impurity removal. The above results show that the walnut protein isolates obtained by the present application have a more excellent potential for improving memory impairment activity.

[0113] According to statistics, the elderly are the main group of people with memory impairment. Therefore, it is of great significance to characterize the improved memory capacity of the walnut protein isolates prepared after digestion by the elderly. Figure 6 Shown is the in vitro digestibility of the example walnut protein isolates A-C and the comparative example walnut protein isolates 1-3 under a simulated old person digestion model. After significant analysis of the results, it was found that the in vitro digestibility of the three example walnut protein isolates A-C was at a relatively high level. The digestibility of the comparative example 1 walnut protein was relatively low, because the residual AOT affected the digestion effect of the gastrointestinal digestive enzymes on the walnut protein. As is known, the nutritional value of protein is closely related to its digestibility, so it can be seen that the three example walnut protein isolates can produce a stronger nutritional value after oral administration.

[0114] Figure 7 Shown is the AChE inhibitory activity of the example and the comparative example walnut protein isolates after in vitro gastrointestinal digestion. Similar to the results of the in vitro digestion experiment, the three example walnut protein isolates A-C have stronger AChE inhibitory activity, which may have the following two reasons: (1) higher in vitro digestibility means that the three example walnut protein isolates can be broken down into more small molecule peptides, thereby increasing the probability of release of AChE inhibitory peptides; (2) the arginine content of the example walnut protein isolates 1-3 is relatively high (Table 2), so there may be more peptides with AChE inhibitory activity in the released small molecule peptides.

[0115] In addition, by Figure 8 and Figure 9 Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 It can be seen that the walnut protein isolates of the three examples exhibit strong antioxidant properties, especially ABTS scavenging ability. This may be because the walnut protein isolates of the examples release more antioxidant peptides during the digestion process. However, it is worth noting that there is no significant difference in DPPH scavenging ability between the walnut protein isolates A to C obtained in the three examples and the walnut protein of comparative example 3. This is mainly because the walnut protein of comparative example 3 exposes more enzyme cleavage sites due to structural expansion during the extraction process, and produces more DPPH scavenging peptides after gastrointestinal digestion. The results show that the walnut protein isolate prepared by the present invention has the potential to improve memory impairment in the elderly after oral administration.

[0116] In summary, the three examples of walnut protein isolates 1 to 3 have the most excellent apparent properties, including the highest solubility, foaming stability, emulsification stability, arginine content, in vitro digestibility in elderly models, AChE inhibition rate after digestion, and antioxidant capacity, and have low surfactant residues. Therefore, the walnut protein isolate preparation method of the present invention is an ideal solution for improving memory impairment in the elderly, having high-quality processing characteristics, and being suitable for large-scale industrial production.

[0117] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for preparing walnut protein isolate, characterized by It comprises the following steps: (1) dissolving sodium bis(2-ethylhexyl) succinate sulfonate in n-hexane to prepare solution A, then mixing a buffer solution containing 0.05-0.1 M KCl with solution A to form a reverse micelle solution, and then mixing defatted walnut powder with the reverse micelle solution, stirring to make it uniform, centrifuging, and collecting the supernatant to obtain walnut protein extract 1; (2) mixing walnut protein extract 1 with a buffer solution containing 0.8-1.0 M KCl, stirring to make it uniform, centrifuging, and then standing to separate the layers, collecting the middle layer and the lower aqueous phase, mixing, and obtaining walnut protein extract 2; (3) adjusting the pH value of walnut protein extract 2 to 4.5, standing, centrifuging, collecting the upper layer and the precipitate, mixing, and obtaining walnut protein isolate 3; adding 0.1-0.5 mM NaCl in anhydrous ethanol as a solvent to make the final concentration reach 0.01-0.03 mM to prepare solution B; mixing walnut protein isolate 3 with solution B, stirring to make it uniform, standing, centrifuging, and collecting the precipitate to obtain walnut protein isolate 4; (4) adding deionized water to walnut protein isolate 4, stirring to make it uniform, then separating through an ultrafiltration membrane with a membrane flux of 10000 Da, collecting the retentate, and freeze-drying to obtain walnut protein isolate.

2. The method of claim 1, wherein: The buffer solution and solution A in step (1) are mixed at a volume ratio of 1:(18-22).

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

4. The method of claim 1, wherein: The walnut protein extract 1 and the buffer solution in step (2) are mixed at a volume ratio of 1:(1-2).

5. The method of claim 1, wherein: The walnut protein isolate 3 and solution B in step (3) are mixed at a ratio of 1:(20-30) (w / v).

6. A walnut protein isolate, characterized in that The walnut protein isolate is prepared by the method of any one of claims 1-5.

7. The walnut protein isolate of claim 6 for use in the preparation of a medicament or health food for assisting in improving memory.

8. The walnut protein isolate of claim 6 for use in the preparation of a medicament or health food for assisting in improving memory of the elderly.

9. The walnut protein isolate of claim 6 for use in the preparation of an acidic food.

10. The walnut protein isolate of claim 6 for use in the preparation of an emulsified food or a foamed food.

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