Preparation method and application of novel walnut intelligence-improving peptide

Abstract

The invention relates to the technical field of walnut protein peptide modification, in particular to preparation and application of novel walnut intelligence-improving peptide, which mainly utilizes polyphenol-mediated Maillard covalent modification to weaken the bitter taste of walnut protein peptide, and controls the addition amount, reaction temperature, reaction time, pH and other external conditions of the walnut protein peptide to obtain the novel walnut intelligence-improving peptide. Screening the walnut protein intelligence-improving peptide by using indexes such as sensory evaluation and anti-oxidation characteristics; then, the influence of the modified walnut protein peptide on D-galactose induced brain injury model mice is deeply explored. Finally, the polyphenol-mediated Maillard reaction covalently modified walnut protein peptide powder is prepared by virtue of the technology, and the ingredient greatly increases the application of the walnut protein peptide in brain-strengthening and intelligence-improving products for alleviating brain injury and the like. The bitter taste of the walnut protein peptide obtained by the method is greatly reduced, and the walnut protein peptide has a potential brain protection effect.

Description

Technical Field

[0001] The invention relates to the technical field of protein peptides, and in particular to a preparation method and application of a novel walnut intelligence-enhancing peptide. Background Art

[0002] Walnut protein is a high-quality protein source with high nutritional value. It is rich in essential amino acids and a variety of bioactive peptides that are beneficial to health. Walnut protein can be converted into walnut protein peptides through specific enzymatic hydrolysis technology. These peptides show significant biological activity in anti-oxidation, cardiovascular protection, and memory improvement. However, walnut protein peptides often encounter bitterness problems during application. This may be due to the exposure of hydrophobic amino acids originally wrapped in walnut protein. These hydrophobic amino acids can stimulate taste buds and produce bitterness. The more hydrophobic amino acids are exposed, the stronger the bitterness. Studies have also shown that when hydrophobic amino acid residues are located at the C-terminus or N-terminus of the polypeptide chain, bitterness will be produced, and the hydrophobic amino acids at the C-terminus are more likely to cause bitterness than those at the N-terminus. Therefore, the bitterness of walnut protein peptides limits its application in the food industry. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a preparation method and application of a novel walnut peptide for improving intelligence, which mainly utilizes the covalent modification effect and molecular polarization effect of the Maillard reaction to increase the degree of freedom of the protein peptide, promote the structure of the walnut protein peptide, fully expose and destroy the bitter region of the walnut protein peptide, and help increase the combination of polysaccharides and the bitter region; on this basis, the Maillard reaction process is regulated and the reaction product is controlled by tea polyphenols, and then the reaction process is terminated by gradient cooling and sudden temperature change technology. Subsequently, the unreacted walnut protein peptide and polysaccharide are removed and the Maillard reaction product is concentrated by ultrafiltration dialysis and other technologies. The bitterness of the walnut protein prepared by the present invention is significantly reduced, and the ability to alleviate brain damage is high, and it can be used as a high-end ingredient to be added to health foods and medicines for strengthening the brain and improving intelligence. The entire preparation process is green and environmentally friendly, low in cost and conducive to large-scale production. In order to solve the above technical problems, the technical solution adopted by the present invention is: A method for preparing a novel walnut intelligence-enhancing peptide comprises the following steps: (1) Pretreatment of walnuts: After removing the walnut shells, the walnut shells are removed by water immersion, and then the shells are removed and dried in an oven until completely dry; (2) The pretreated walnut kernels are subjected to low temperature oil pressing to remove oil, and the remaining oil is removed by petroleum ether from the obtained walnut cake, and the petroleum ether is removed by suction filtration, and then the treated cake is dried and ground into fine powder; (3) Enzymatic hydrolysis of walnut protein meal: adding enzyme to walnut protein meal for enzymatic hydrolysis to obtain enzymatic hydrolyzate; (4) Inactivating the enzyme: inactivating the enzymatic hydrolysate obtained in (3), and centrifuging to obtain the supernatant; (5) Primary ultrafiltration: The supernatant treated in (4) is ultrafiltered twice, and water is added for concentration to obtain a crude walnut protein peptide solution; (6) Vacuum freeze drying: freeze-drying the concentrated enzymatic hydrolysis solution to obtain walnut protein peptide; (7) Maillard reaction dry basis stage: grind and mix the walnut protein peptide in (6) with the sugar substance, and then react; (8) Maillard reaction wet-base stage: add water to the reaction mixture in (7) to fully dissolve it, and then react in an oil bath at a certain temperature; (9) Terminating the reaction process: The Maillard reaction solution obtained in (8) is terminated by a gradient cooling-sudden temperature control method; (10) Ultrafiltration: The Maillard reaction solution obtained in step (9) is subjected to ultrafiltration; (11) Spray drying: The reaction solution treated in (10) is freeze-dried to obtain a walnut protein peptide with improved bitterness.

[0004] Furthermore, in the step (1), the mass ratio of walnut kernel to water is 1: (10-50), the stirring rate is 100-200 r / min, the time is 1-2 h, the water temperature is 40-60 ° C; the drying temperature is 45-60 ° C, and the drying time is 30-36 h; In the step (2), the low-temperature oil pressing temperature is 50-60°C, and the conditions for removing the remaining oil with petroleum ether are: the mass ratio of walnut meal to petroleum ether is 1: (5-20), the stirring rate is 300-400 r / min, the extraction is 10-12 h, and the extraction process is repeated 2-4 times. Furthermore, in step (3), the enzyme is papain or flavor protease or alkaline protease or neutral protease or trypsin; the enzyme activity is 6000-8000 U / mg, the mass ratio of enzyme to substrate is: 1:(50-200), and the conditions for different enzymolysis are: the hydrolysis temperature of papain is 50-60°C, and the pH is 6-7; the hydrolysis temperature of flavor protease is 50-60°C, and the pH is 6-7; the hydrolysis temperature of alkaline protease is 40-60°C, and the pH is 8-10; the hydrolysis temperature of neutral protease is 45-65°C, and the pH is 6.0-7.0; the hydrolysis temperature of trypsin is 35-45°C, and the pH is 7-9; the hydrolysis time is 3-5 h, preferably, the hydrolysis time is 3-4 h; In the step (4), the enzyme is inactivated by treatment at 80-95°C for 15-25 min, and the centrifugation condition is 5000-7000 r / min.

[0005] Furthermore, in step (5), ultrafiltration is performed using an ultrafiltration membrane of 1-3 kDa: preferably, the ultrafiltration membrane is 1 kDa or 2 kDa; In step (6), the freeze-drying conditions are temperature of -50~-40 °C, vacuum degree of 0.1~0.2 Pa, and time of 32-38 h.

[0006] Furthermore, in step (7), the sugar is glucose, fructose, sucrose or astragalus polysaccharide, and the mass ratio of walnut protein peptide to sugar is (1-3): (1-3); the conditions of the Maillard reaction dry stage are that the walnut protein peptide and sugar are ground and mixed, placed at 70-90 ° C, and reacted for 30-40 min; Furthermore, in step (8), the conditions of the Maillard reaction wet-base stage are: the center temperature of the solution is 90-110°C, and the treatment is 40-60 min.

[0007] Furthermore, in step (8), polyphenols including tea polyphenols, EGCG, chlorogenic acid, quercetin and the like are added during the reaction, and the amount of polyphenols added is 1% to 4% of the walnut protein peptide. The bitterness of the reaction product and the reaction intensity are used as evaluation indicators, and tea polyphenols are preferred.

[0008] Furthermore, in step (9), the termination reaction conditions are as follows: in the early stage, the temperature is cooled by gradient cooling, the cooling rate is 10°C / min, the gradient cooling time is 3-5 min, and then the temperature is rapidly cooled in an ice water bath (about 0°C) for 10-15 min.

[0009] Furthermore, in step (10), the supernatant in step (10) is diluted with water and then ultrafiltered using a 40-60 kDa ultrafiltration membrane. After the ultrafiltration is completed, water is added to completely reflux the Maillard reaction products remaining between the collection pipeline and the ultrafiltration membrane; preferably, the ultrafiltration membrane is 50 kDa or 60 kDa; In step (11), the spray drying conditions are: blower temperature: 50-60 °C, atomization temperature: 170-180 °C, air outlet temperature: 80-90 °C, fan setting: 55-60 Hz, peristaltic pump speed: 11-12 m / s.

[0010] The novel walnut intelligence-enhancing peptide obtained by the preparation method of the novel walnut intelligence-enhancing peptide is used as a health food or medicine for alleviating brain damage and strengthening the brain and improving intelligence.

[0011] The present invention has the following beneficial effects: 1. The present invention covalently regulates walnut protein peptides by means of the Maillard reaction, exposes bitter amino acids in walnut protein peptides, increases the probability of binding of polysaccharides and protein peptides with bitter amino acids, and thus reduces the bitterness of walnut protein peptides. 2. The present invention regulates the Maillard reaction by means of polyphenol mediation, regulates the degree of binding between walnut protein peptides and polysaccharides, and then controls the process of the Maillard reaction, thereby avoiding the generation of macromolecular substances in the reaction, effectively improving the utilization rate of walnut protein peptides and polysaccharides, and is conducive to improving the flavor of walnut protein peptides and promoting the development of related research and industries.

[0012] 3. The present invention uses the Maillard reaction mediated by polyphenols, and its product not only has the effect of reducing bitterness, but also has the effect of reducing brain damage, thereby greatly improving the biological activity function of walnut protein peptides, thereby further broadening the industrial application of walnut protein peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The effect of different reaction intensities on the Maillard reaction intensity; Figure 2 The effect of different reaction intensities on the content of AGEs in the Maillard reaction; Figure 3 Sensory evaluation of walnut protein peptides by Maillard reaction; Figure 4 Effects of Maillard reaction products of walnut protein peptides on brain-injured mice; Figure 5 Effects of Maillard products of walnut protein peptides on histopathology of hippocampus in brain-injured mice. DETAILED DESCRIPTION

[0014] The embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0015] The comparative measurement methods and results of the above examples are as follows: 1. Determination of Maillard reaction intensity Take 3 mL of the reaction solution after centrifugation (the reaction solution after step (8) in Example 1 is centrifuged), dilute it 10 times, and measure its absorbance at a wavelength of 420 nm. The result is expressed as A420 nm.

[0016] 2. Determination of AGEs 1 g of walnut protein peptide Maillard reaction product was added to 19 mL of phosphate buffer solution (0.05 mol / L, pH 7.4). The fluorescence intensity was measured using a fluorescence spectrophotometer (F-2700, HITACHI, Shanghai, China). Excitation wavelength: 345 nm. Emission wavelength: 425 nm. Width: 5 nm. The results were expressed in AU / g. The calculation formula is as follows: Fluorescent AGEs content = FI / m (AU / g) Where FI represents the fluorescence intensity of the sample, and m represents the weight of the sample.

[0017] 3. Sensory evaluation The evaluation team was composed of 10 graduate students majoring in food. They were uniformly trained before the evaluation and conducted sensory evaluation on the flavor and taste of the walnut protein peptides produced by the Maillard reaction according to Table 1.

[0018] Table 1 Sensory evaluation table

[0019] 4. Determination of bitterness intensity An 11-member sensory evaluation panel, aged between 25 and 40, including 5 women and 6 men, none of whom had known congenital taste defects, was tasked with evaluating the bitterness of the samples. For this evaluation, they used three different concentrations of quinine hydrochloride solution as a reference standard for bitterness: 2 mg / L, 5 mg / L, and 10 mg / L, corresponding to a bitterness score of 2, 5, and 8, respectively.

[0020] 5. Animal experiments Male ICR mice used in this study were purchased from Hefei University of Technology (Hefei, China), including 90 young (6 weeks old, weighing 24 ± 1 g) mice. The animals were housed in metabolic cages in a sterile animal room under constant conditions (25 ± 2 °C, 60% ± 10% relative humidity) with alternating 12-h light / dark cycles. During the experiments, the mice had ad libitum access to commercial food and laboratory sterile water. Animal management strictly complied with the regulations for the care and use of experimental animals approved by the Animal Experimental Ethics Committee of Hefei University of Technology (Hefei, China).

[0021] After one week of adaptive feeding (denoted as 0 week), ICR mice were randomly divided into the following 9 groups, with 10 mice in each group: normal control (NC), D-galactose-induced aging group (MC), glutathione (GSH), walnut protein peptide (WPP), Maillard reaction walnut protein peptide (WA MPs), polyphenol-regulated Maillard reaction walnut protein peptide (WPA MPs), Maillard reaction walnut protein peptide before dry basis reaction (WPA MPs-A), Maillard reaction walnut protein peptide after wet basis reaction (WPA MPs-L), and Maillard reaction walnut protein peptide with changed dry basis condition (WPA MPs-T). The mice in the NC group did not receive any treatment, but were given food, water and an equal amount of normal saline. The mice in the MC, WPP, W-AMPs and WA MPs groups were intraperitoneally injected with d-galactose (500 mg / kg / d) at 9:00 AM, respectively. Each group was then given the corresponding substances by gavage at a dose of 400 mg / kg / day for 6 weeks. During the experiment, the body weight and food intake of the mice were monitored at a fixed time every week. After completing the feeding procedure, all mice were fasted and given water for 12 h, and then euthanized using carbon dioxide gas. Blood from the heart was then collected after centrifugation at 4500 rpm for 15 minutes at 4 °C to separate serum.

[0022] 6. Determination of acetylcholinesterase (AChE): The AChE activity in mouse brain tissue was determined using the AChE Nanjing Jiancheng Kit.

[0023] 7. Determination of Choline acetyltransferase (ChAT): The activity of ChAT in mouse brain tissue was determined using the ChAT Nanjing Jiancheng Kit.

[0024] Example 1 1) Pretreatment of walnuts: After removing the walnut shells, the walnut shells were removed by soaking in 20 times the volume of water, and then dried in an oven until completely dry.

[0025] 2) Degreasing of walnuts: Use an oil press in low-temperature oil pressing mode to remove a large amount of oil, add 10 times the volume of petroleum ether to the walnut cake to remove the remaining oil, extract for 12 hours, repeat the extraction process 3 times, filter to remove the petroleum ether, remove a large amount of lipids in the walnut cake, and then dry the cake and grind it into fine powder; 3) Enzymatic hydrolysis of walnut protein: Under the condition of 6000 U / mg enzyme activity, the mass ratio of papain to substrate was 1:80; the enzymatic hydrolysis temperature was 50 ℃, the pH was 7; and the hydrolysis time was 3 h.

[0026] 4) Inactivation of enzymes: The enzymatic hydrolysate was treated at 90 °C for 20 min to inactivate the enzymes, and then centrifuged at 6000 r / min to obtain the supernatant.

[0027] 5) Primary ultrafiltration: The supernatant is ultrafiltered twice, and water is added for concentration to obtain a crude walnut protein peptide solution; 6) Vacuum freeze drying: The concentrated ovotransferrin solution was placed in a freeze drying box and placed in a -20 ℃ refrigerator for pre-freezing, and then placed in a freeze dryer for freeze drying. The freeze drying conditions were -50 ℃, vacuum degree 0.1 Pa, and time for 36 h.

[0028] 7) Maillard reaction (dry basis): the walnut protein peptide in (6) and glucose were fully ground and mixed at a mass ratio of 1:2, and then placed at 70 °C for 15 min; Based on (8), polyphenols were added before the reaction to regulate the Maillard reaction product of walnut protein peptide; the amount of polyphenols added was 2% of the mass of walnut protein peptide. 8) Maillard reaction (wet basis): Add 50 mL of deionized water to the mixture in (7) and perform Maillard reaction at 100 °C; 9) Termination of reaction process: The Maillard reaction solution in (8) is terminated by gradient cooling-sudden temperature control method. In the early stage, the temperature is cooled by gradient cooling, the cooling rate is 10 ℃ / min, and the gradient cooling time is 3-5 min. Then, it is placed in an ice water bath (about 0 ℃) for rapid cooling, and the standing time is: 10-15 min.

[0029] 10) Ultrafiltration: After diluting the supernatant in (9) with water, use an ultrafiltration membrane for ultrafiltration. After the ultrafiltration is completed, add water to completely reflux and collect the Maillard reaction products remaining between the pipeline and the ultrafiltration membrane; the ultrafiltration membrane is 50 kDa; 11) Spray drying: freeze-dry the reaction solution treated in (10) to obtain a bitterness-improved walnut protein peptide nootropic ingredient. The spray drying conditions are: blower temperature: 55 °C, atomization temperature: 175 °C, air outlet temperature: 85 °C, fan setting: 60 Hz, peristaltic pump speed: 11 m / s.

[0030] Optimization of polyphenol addition Based on Example 1, 9) different polyphenol addition amounts were changed, the polyphenol was tea polyphenol, and according to the reaction intensity and bitterness evaluation, preferably, the polyphenol addition amount was selected to be 2% of the walnut protein peptide, and the other conditions were the same as in Example 1. Figure 1-3. The untreated walnut protein peptide has a significant bitter taste and an unacceptable aftertaste. After the flavor of the walnut protein peptide was covalently modified by the Maillard reaction, the sensory evaluation of each embodiment changed significantly. Compared with the groups without polyphenols, the reaction was lowest when the reaction temperature was low and the reaction time was short (Example 1). After polyphenols were added, the degree of Maillard reaction changed with the change of polyphenol concentration. When the concentration was lower than 2%, the addition of polyphenols significantly inhibited the degree of Maillard reaction; when the concentration was higher than 2%, the addition of polyphenols significantly promoted the degree of Maillard reaction.

[0031] Compared with the groups without polyphenols, the reaction was lowest when the reaction temperature was low and the reaction time was short (Example 1). After polyphenols were added, the Maillard reaction product AGEs changed with the change of polyphenol concentration. When the concentration was lower than 2%, the addition of polyphenols significantly inhibited the content of AGEs in the Maillard reaction product; when the concentration was higher than 2%, the addition of polyphenols significantly promoted the content of AGEs in the Maillard reaction product.

[0032] Example 2 Based on Example 1, the difference is 7) Maillard reaction (7): walnut protein peptide and glucose 2:1, and the rest is the same as Example 1.

[0033] Example 3 Based on Example 1, the difference is 7), Maillard reaction (7): the ratio of walnut protein peptide to glucose is 1:3, and the rest is the same as Example 1.

[0034] Example 4 Based on Example 1, the difference is 7), Maillard reaction (7): the ratio of walnut protein peptide to astragalus polysaccharide is 1:1, and the rest is the same as Example 1.

[0035] Example 5 Based on Example 1, the difference is 7), Maillard reaction (7): the ratio of walnut protein peptide to astragalus polysaccharide is 1:2, and the rest is the same as Example 1.

[0036] Example 6 Based on Example 1, the difference is 7) Maillard reaction (7): the reaction temperature of walnut protein peptide and glucose is 90°C, and the rest is the same as Example 1.

[0037] Example 7 Based on Example 1, the difference is 7) Maillard reaction (7): the reaction temperature of walnut protein peptide and glucose is 100°C, and the rest is the same as Example 1.

[0038] Example 8 Based on Example 1, the difference is 7) Maillard reaction (7): the reaction temperature of walnut protein peptide and glucose is 110°C, and the rest is the same as Example 1.

[0039] Example 9 Based on Example 1, the difference is 7) Maillard reaction (7): the reaction temperature of walnut protein peptide and glucose is 120°C, and the rest is the same as Example 1.

[0040] Example 10 Based on Example 1, the difference is 7) Maillard reaction (7): the reaction time of walnut protein peptide and glucose is 60 min.

[0041] Embodiment 11 Based on Example 1, the difference is 7) Maillard reaction (7): the reaction time of walnut protein peptide and glucose is 80 min.

[0042] Example 12 Based on Example 1, the difference is 7) Maillard reaction (7): the reaction time of walnut protein peptide and glucose is 100 min.

[0043] Embodiment 13 Based on Example 1, the difference is 7) Maillard reaction (7): the reaction time of walnut protein peptide and glucose is 110 min.

[0044] Comparative Example 1 1) Pretreatment of walnuts: After removing the shell of the walnuts, the kernel seed coat is removed by soaking in 20 times the volume of water, and then the walnuts are dried in an oven until completely dry.

[0045] 2) Degreasing of walnuts: Use an oil press in low-temperature oil pressing mode to remove a large amount of oil, add 10 times the volume of petroleum ether to remove the remaining oil from the walnut cake, extract for 12 hours, repeat the extraction process 2-4 times, filter to remove the petroleum ether, remove a large amount of lipids in the walnut cake, and then dry the cake and grind it into fine powder; 3) Enzymatic hydrolysis of walnut protein: Under the condition of 6000 U / mg enzyme activity, the mass ratio of papain to substrate was 1:80; the enzymatic hydrolysis temperature was 50 ℃, the pH was 7; and the hydrolysis time was 3 h.

[0046] 4) Inactivation of enzymes: The enzymatic hydrolysate was treated at 90 °C for 20 min to inactivate the enzymes, and then centrifuged at 6000 r / min to obtain the supernatant.

[0047] 5) Primary ultrafiltration: The supernatant is ultrafiltered twice, and water is added for concentration to obtain a crude walnut protein peptide solution; 6) Vacuum freeze drying: The concentrated walnut protein peptide solution was placed in a freeze drying box and placed in a -20 ℃ refrigerator for pre-freezing, and then placed in a freeze dryer for freeze drying. The freeze drying conditions were -50 ℃, vacuum degree 0.1 Pa, and time for 36 h.

[0048] Comparative Example 2 The difference from Example 1 is that step 7) is not performed, and the rest is the same as Example 1.

[0049] Comparative Example 3 The difference from Example 1 is that tea polyphenols are added before the reaction in step 7), and the rest is the same as Example 1.

[0050] Comparative Example 4 The difference from Example 1 is that tea polyphenols are added after the reaction in step 8), and then the Maillard termination reaction is carried out in step 9). Other points are the same as in Example 1.

[0051] Comparative Example 5 The difference from Example 1 is that in step 7), the Maillard reaction temperature is 80° C. and the reaction is carried out for 35 min.

[0052] By controlling the reaction conditions such as the amount of polysaccharide added (Examples 2-3), reaction time (Examples 4-5), and reaction temperature (Examples 6-7), the sensory evaluation changed significantly, among which Example 7 and Example 2 had lower astringency. The effect of Maillard reaction modification on the bitterness of walnut protein peptide is shown in Table 2. As can be seen from Table 2, Maillard reaction modification significantly improved the bitterness of walnut protein peptide, but the influence of reaction conditions is crucial to the bitterness regulation of walnut protein peptide. Compared with Comparative Example 1, under a certain reaction intensity (such as Examples 2 to 4), the bitterness of walnut protein peptide modified by Maillard reaction decreased significantly. However, with the extension of reaction time and the increase of reaction temperature (as shown in Examples 5 to 6), the bitterness increased. After adding polyphenols, it not only has the effect of enhancing bitterness inhibition, but also has the effect of delaying the bitterness degree under the same Maillard reaction intensity (Examples 7 to 8). Compared with the group (Comparative Example 2) that did not undergo Maillard reaction under dry conditions, the bitterness degree of Maillard dry-wet mixed reaction (Example 1) was significantly improved. In addition, the addition time of polyphenols can also be used as one of the key factors for regulating the bitterness of walnut protein peptides. The bitterness thresholds of polyphenols at different reaction stages (addition before dry-based reaction (Comparative Example 3), addition before wet-based reaction (Example 1), and addition after wet-based reaction (Comparative Example 4)) also have significant differences, among which the bitterness threshold of Example 1 is the lowest. In addition, the dry-based reaction temperature and time also significantly affect its bitterness. Compared with Example 1, the bitterness is significantly reduced when the dry-based Maillard reaction temperature increases and the reaction time is prolonged (Comparative Example 5). Therefore, the bitterness of walnut protein peptides after the combination of dry-wet Maillard reaction and polyphenol-mediated regulation is significantly improved. This may be because the bitter hydrophobic amino acids or hydrophobic regions in the walnut protein peptide participate in the Maillard reaction and generate new flavor characteristics that may have sweet, fruity or other pleasant flavors. These flavor characteristics can mask or balance the bitterness, thereby improving the overall taste. In addition, in the Maillard dry-base reaction, walnut protein peptides and polysaccharides interact with each other through non-covalent interactions such as hydrogen bonds and electrostatic interactions, combined with the control of water activity to control the reaction process and product formation, and ultimately improve the bitterness of walnut protein peptides. During the Maillard reaction, HMF (5-hydroxymethylfurfural) is more easily generated under wet heat conditions; DDMP (2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one) is more easily generated under dry heat conditions. At lower water activity, the reaction rate slows down, which can reduce the formation of bitter substances. By controlling processing conditions such as temperature, time and moisture content, the formation of unpleasant bitter compounds can be reduced. Polyphenols can competitively regulate the generation of bitter substances through antioxidant activity and the release of oxygen atoms, and ultimately improve the bitterness of walnut protein peptides.

[0053] Table 2 Sensory evaluation table Group Bitterness rating (with quinine as reference) Example 1 4.31 Example 2 7.22 Example 3 7.47 Example 4 7.94 Example 5 7.36 Example 6 6.4 Example 7 4.54 Example 8 7.61 Example 9 5.46 Example 10 5.84 Embodiment 11 5.76 Example 12 5.23 Embodiment 13 5.34 Comparative Example 1 8.95 Comparative Example 2 5.62 Comparative Example 3 6.95 Comparative Example 4 5.92 Comparative Example 5 7.03 Effects of Maillard reaction products of walnut protein peptide on brain-damaged mice Effects of Maillard reaction products of walnut protein peptides on brain-damaged mice Figure 4 As shown. Figure 4 It can be seen that compared with the D-galactose-induced brain injury model group (MC group), the acetylcholinesterase (AChE) activity of brain tissue of brain-injured mice given the Maillard reaction-modified walnut protein peptide (Comparative Example 2) group (WA MPs) and the polyphenol-mediated walnut protein peptide Maillard reaction product (Example 1) group (WPA MPs) was significantly reduced, while the choline acetyltransferase (ChAT) enzyme activity was significantly increased. In addition, compared with the untreated walnut protein peptide (WPP, i.e., Comparative Example 1), after the product prepared in Comparative Example 1 was administered to the injured mice, the AChE activity of the brain tissue was significantly reduced and the ChAT activity was significant, while the improvement of the above enzymes in the polyphenol-regulated walnut protein Maillard reaction product (Example 1) group was more significant. The Maillard reaction products under polyphenol differential mediation (WPA MPs-A, i.e., Comparative Example 3, WPA MPs-L, i.e., Comparative Example 4) and dry-base reaction conditions (WPA MPs-T, i.e., Comparative Example 5) all had certain alleviating and improving effects on brain-injured mice, but the effect of Example 1 was significantly better than that of the above comparative examples. Effects of Maillard reaction products of walnut protein peptides on histopathology of hippocampus in brain-injured mice The hippocampus is a key area in the brain that is closely related to learning and memory functions, especially in the formation of spatial memory and long-term memory. Through pathological observation of mouse hippocampal tissue, researchers can evaluate the extent and nature of brain damage, including neuronal damage, apoptosis, synaptic changes, and glial cell responses. The effect of the Maillard reaction products of walnut protein peptides on the pathology of hippocampal tissue in brain-damaged mice Figure 5 (Shown. Figure 5 It can be seen that compared with the normal group (i.e. Figure 5Compared with the NC group, the other groups all showed cytoplasmic vacuolation, and some cells showed nuclear fission and fragmentation, among which the phenomenon of the brain injury model (i.e., MC) group was the most significant. In addition, the number of apoptotic cells identified by shrunken cells, concentrated cytoplasm and pus cell nuclei in the MC group was the largest, indicating that the degree of damage and necrotic cells in the MC group reached the maximum. After taking walnut protein peptide (WPP, i.e., prepared in Comparative Example 1), Maillard walnut protein peptide (WA MPs, i.e., the product obtained in Comparative Example 2) and polyphenol-regulated Maillard walnut protein peptide (WPA MPs, i.e., the product obtained in Example 1), hippocampal vacuolation was significantly reversed, especially the reversal effect of WPA MPs was more significant. The Maillard reaction products under polyphenol differential mediation (WPA MPs-A, i.e., Comparative Example 3, WPA MPs-L, i.e., Comparative Example 4) and dry-base reaction conditions (WPA MPs-T, i.e., Comparative Example 5) all had a certain alleviation and improvement effect on hippocampal vacuolation in brain-injured mice, but the effect of Example 1 was significantly better than that of the above-mentioned comparative examples.

[0054] The above-mentioned embodiments are only preferred technical solutions of the present invention and should not be regarded as limitations of the present invention. The embodiments and features in the embodiments of the present application can be arbitrarily combined with each other without conflict. The protection scope of the present invention shall be the technical solutions recorded in the claims, including the equivalent replacement solutions of the technical features in the technical solutions recorded in the claims. That is, equivalent replacement improvements within this scope are also within the protection scope of the present invention.

Claims

1. A method for preparing a novel walnut intelligence-enhancing peptide, characterized in that: The following steps are involved: (1) Pretreatment of walnuts: After removing the walnut shells, the walnut shells are removed by water immersion, and then the shells are removed and dried in an oven; (2) The pretreated walnut kernels are subjected to low temperature oil pressing to remove oil, and the remaining oil is removed by petroleum ether from the obtained walnut cake, and the petroleum ether is removed by suction filtration, and then the treated cake is dried and ground into fine powder; (3) Walnut protein enzymatic hydrolysis: Walnut protein is enzymatically hydrolyzed by adding enzymes to obtain enzymatic hydrolyzate; (4) Inactivating the enzyme: inactivating the enzymatic hydrolysate obtained in (3), and centrifuging to obtain the supernatant; (5) Primary ultrafiltration: The supernatant treated in (4) is ultrafiltered twice, and water is added for concentration to obtain a crude walnut protein peptide solution; (6) Vacuum freeze drying: freeze-drying the concentrated enzymatic hydrolysis solution to obtain walnut protein peptide; (7) Maillard reaction dry basis stage: grind and mix the walnut protein peptide in (6) with the sugar substance, and then react; (8) Maillard reaction wet-base stage: add water to the reaction mixture in (7) to fully dissolve it, and then react in an oil bath at a certain temperature; (9) Terminating the reaction process: The Maillard reaction solution obtained in (8) is terminated by a gradient cooling-sudden temperature control method; (10) Ultrafiltration: The Maillard reaction solution obtained in step (9) is subjected to ultrafiltration; (11) Spray drying: The reaction solution treated in (10) is freeze-dried to obtain a walnut protein peptide with improved bitterness.

2. The method for preparing the novel walnut intelligence-enhancing peptide according to claim 1, characterized in that: In the step (1), the mass ratio of walnut kernel to water is 1: (10-50), the stirring rate is 100-200 r / min, the time is 1-2 h, the water temperature is 40-60°C; the drying temperature is 45-60°C, and the drying time is 30-36 h; In the step (2), the low-temperature oil pressing temperature is 50-60°C, the conditions for removing the remaining oil with petroleum ether are: the mass ratio of walnut protein meal to petroleum ether is 1: (5-20), the stirring rate is 300-400 r / min, the extraction is 10-12 h, and the extraction process is repeated 2-4 times.

3. The method for preparing the novel walnut intelligence-enhancing peptide according to claim 1, characterized in that: In the step (3), the enzyme is papain or flavor protease or alkaline protease or neutral protease or trypsin; the enzyme activity is 6000-8000 U / mg, the mass ratio of enzyme to substrate is 1:(50-200), and the conditions for different enzymolysis are: the hydrolysis temperature of papain is 50-60°C and the pH is 6-7; the hydrolysis temperature of flavor protease is 50-60°C and the pH is 6-7; The hydrolysis temperature of alkaline protease is 40-60°C, and the pH is 8-10; the hydrolysis temperature of neutral protease is 45-65°C, and the pH is 6.0-7.0; the hydrolysis temperature of trypsin is 35-45°C, and the pH is 7-9; the hydrolysis time is 3-5 h, preferably, the hydrolysis time is 3-4 h; In the step (4), the enzyme is inactivated by treatment at 80-95°C for 15-25 min, and the centrifugation condition is 5000-7000 r / min.

4. The method for preparing the novel walnut intelligence-enhancing peptide according to claim 1, characterized in that: In the step (5), ultrafiltration is performed using an ultrafiltration membrane of 1-3 kDa: preferably, the ultrafiltration membrane is 1 kDa or 2 kDa; In step (6), the freeze-drying conditions are temperature of -50~-40°C, vacuum degree of 0.1~0.2 Pa, and time of 32-38h.

5. The method for preparing the novel walnut intelligence-enhancing peptide according to claim 1, characterized in that: In step (7), the sugar is glucose, fructose or sucrose, and the mass ratio of walnut protein peptide to sugar is (1-3): (1-3). The conditions of the dry stage of the Maillard reaction are as follows: after the walnut protein peptide and sugar are ground and mixed, they are placed at 70-90 ° C and reacted for 30-40 min.

6. The method for preparing the novel walnut intelligence-enhancing peptide according to claim 1, characterized in that: In step (8), the conditions of the Maillard reaction wet-base stage are: the center temperature of the solution is 90-110°C, and the treatment time is 40-60 min.

7. The method for preparing the novel walnut intelligence-enhancing peptide according to claim 1, characterized in that: In step (8), polyphenols including tea polyphenols, EGCG, chlorogenic acid, quercetin or other polyphenols are also added during the reaction, preferably tea polyphenols; the amount of polyphenols added is 1% to 4% of the mass of the walnut protein peptide, and preferably 2%.

8. The method for preparing the novel walnut intelligence-enhancing peptide according to claim 1, characterized in that: In step (9), the termination reaction conditions are: cooling by gradient cooling in the early stage, and rapidly cooling in an ice water bath; preferably, the gradient cooling rate is 10 °C / min, the gradient cooling time is 3-5 min, and the rapid cooling time in the ice water bath is 10-15 min.

9. The method for preparing the novel walnut intelligence-enhancing peptide according to claim 1, characterized in that: In step (10), the supernatant in step (10) is diluted with water and then ultrafiltered using a 40-60 kDa ultrafiltration membrane. After the ultrafiltration is completed, water is added to completely reflux the Maillard reaction products remaining in the collection pipeline and between the ultrafiltration membranes; preferably, the ultrafiltration membrane is 50 kDa or 60 kDa; In step (11), the spray drying conditions are: blower temperature: 50-60 °C, atomization temperature: 170-180 °C, air outlet temperature: 80-90 °C, fan setting: 55-60 Hz, peristaltic pump speed: 11-12 m / s.

10. The novel walnut intelligence-enhancing peptide obtained by the preparation method of the novel walnut intelligence-enhancing peptide according to any one of claims 1 to 9 is used as a health food or medicine for alleviating brain damage and strengthening the brain and improving intelligence.

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