Walnut polypeptide with sleep promoting function as well as preparation method and application thereof

Abstract

The invention discloses walnut protein peptide powder with a sleep promoting function as well as a preparation method and application of the walnut protein peptide powder. The walnut protein peptide powder prepared by the method has the sleep promoting function.

Description

TECHNICAL FIELD

[0002] The application belongs to the technical field of functional oligopeptides, and particularly relates to a walnut protein peptide powder with sleep-promoting function and a preparation method and application thereof. BACKGROUND

[0004] Sleep disorders are a global public health problem. Long-term insomnia not only affects learning and work efficiency, but is also closely related to neurological, metabolic, immune and cardiovascular diseases. Although existing sleep aids can improve sleep in the short term, long-term use can lead to tolerance and side effects. Therefore, it is of great significance to develop natural and safe food-derived sleep aids. Sleep regulation involves multiple neurotransmitters, among which gamma-aminobutyric acid (GABA) is a key inhibitory neurotransmitter that reduces neuronal excitability by acting on GABA receptors to promote sleep onset and maintenance. Increasing GABA levels or enhancing its receptor function is one of the important mechanisms of food-derived sleep-active peptides.

[0005] Walnut protein is an important nutritional component in walnut kernels, rich in various structural proteins such as albumin, globulin and glutelin, and has excellent amino acid composition and high nutritional value. In recent years, it has been found that enzymatically hydrolyzed walnut protein can release a variety of small molecule active peptides, some of which have significant neuroregulatory functions and can improve sleep quality by affecting the gamma-aminobutyric acid (GABA) system, melatonin synthesis and antioxidant pathways. As a common nut resource, walnut protein is widely available and moderately priced, and has the potential to be developed into a food-derived sleep aid. However, the enzymatic process and conditions have a significant impact on the composition and activity of the peptides, so exploring an optimized enzymatic process to obtain walnut peptides with better sleep-promoting effects has become an important research direction for the high-value utilization of walnut protein and the development of functional foods. SUMMARY

[0007] The first object of the present application is to provide a preparation method of a walnut protein peptide powder with sleep-promoting function.

[0008] To achieve the above technical purposes, the technical scheme adopted by the present application is as follows:

[0009] A preparation method of a walnut protein peptide powder with sleep-promoting function, comprising:

[0010] Dissolve the walnut protein powder in water, and then add enzymes for enzymatic hydrolysis after ultrasonic treatment of the dissolved solution. After the enzymatic hydrolysis is completed, the added enzymes are inactivated, and the supernatant obtained by centrifugation is subjected to ultrafiltration treatment. The ultrafiltrate obtained is dried into a powder, which is the walnut protein peptide powder.

[0011] The enzyme is selected from one or more of neutral protease, flavour protease, protease, papain, pepsin, alkaline protease, trypsin, preferably protease, flavour protease, pepsin or alkaline protease.

[0012] In some embodiments,

[0013] Before adding the neutral protease, the dissolution solution is adjusted to pH 6.5-7.5 and the temperature is adjusted to 45-55°C.

[0014] Before adding the flavour protease, the dissolution solution is adjusted to pH 7.0-8.0 and the temperature is adjusted to 45-55°C.

[0015] Before adding the protease, the dissolution solution is adjusted to pH 7.0-8.0 and the temperature is adjusted to 45-55°C.

[0016] Before adding the papain, the dissolution solution is adjusted to pH 6.0-7.0 and the temperature is adjusted to 55-65°C.

[0017] Before adding the pepsin, the dissolution solution is adjusted to pH 2.0-3.0 and the temperature is adjusted to 35-40°C.

[0018] Before adding the alkaline protease, the dissolution solution is adjusted to pH 8.5-9.5 and the temperature is adjusted to 45-55°C.

[0019] Before adding the trypsin, the dissolution solution is adjusted to pH 6.5-7.5 and the temperature is adjusted to 35-40°C.

[0020] In some embodiments, the enzyme is selected from a combination of pepsin and papain or a combination of pepsin and flavour protease.

[0021] In some embodiments, before adding the pepsin, the pH value of the mixed solution is adjusted to 2.0-2.5, the temperature is adjusted to 37-40°C, the pepsin is added, and the enzymolysis is performed for 2.5-3 h; the pH value of the mixed solution is adjusted to 7.5-8.0, the temperature is adjusted to 50-55°C, the flavour protease is added, and the enzymolysis is performed for 2.5-3 h.

[0022] In some embodiments, before adding the pepsin, the pH value of the mixed solution is adjusted to 2.0-2.5, the temperature is adjusted to 37-40°C, the pepsin is added, and the enzymolysis is performed for 2.5-3 h; the pH value of the mixed solution is adjusted to 6.5-7.0, the temperature is adjusted to 55-60°C, the papain is added, and the enzymolysis is performed for 2.5-3 h.

[0023] In some embodiments, the enzyme activity of the neutral protease, flavour protease, protease, papain, pepsin, alkaline protease or trypsin is 5500-6500 U / g, preferably 6000 U / g.

[0024] In some embodiments, after the walnut protein powder is added with water to form a mixed solution, the mixed solution is first stirred by magnetic force at room temperature, and then heated in a water bath to completely dissolve the walnut protein powder. Preferably, the preparation method of the walnut protein powder is as follows: using wild walnuts in Xinjiang, the walnuts are broken, ground and defatted to make the protein content reach above 80%.

[0025] In some embodiments, the ultrasonic treatment time is 10-15 min.

[0026] In some embodiments, the pH of the dissolved solution is adjusted by adding hydrochloric acid or sodium hydroxide.

[0027] In some embodiments, the dissolved solution after enzymolysis is placed in boiling water for 10-15 min to inactivate.

[0028] In some embodiments, the ultrafiltration treatment has a molecular weight cut-off of ≤5 kDa, and the enzymolysis solution with a molecular weight of ≤5 kDa is collected.

[0029] The second object of the present application is to provide a walnut protein peptide powder prepared by the above preparation method.

[0030] The third object of the present application is to provide the use of the walnut protein peptide powder prepared by the above preparation method in the preparation of sleep-aiding functional products; the functional products include food and health products.

[0031] The preparation method of the sleep-aiding walnut protein peptide provided by the present application can prepare a sleep-aiding walnut protein peptide powder with effective sleep-aiding effect. The sleep-aiding walnut protein peptide powder prepared by the preparation method of the present application can promote the activity of γ-aminobutyric acid (GABA) and play a calming and sleep-promoting role, and on the other hand, the antioxidant properties of the sleep-aiding walnut protein peptide powder can inhibit the generation of oxidized protein products (OPA) and reduce the damage of oxidative stress to neurons, thereby indirectly improving sleep quality. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 GABA standard curve.

[0034] Figure 2 GABA content of 0.1 mg / mL walnut peptide protein peptides digested by different single enzymes.

[0035] Figure 3 GABA content of 0.1 mg / mL walnut peptide protein peptides digested by different single enzymes.

[0036] Figure 4 ABTS inhibition rate (%) of 0.1 mg / mL single-enzyme-digested walnut peptides.

[0037] Figure 5Inhibition rate of walnut peptide hydrolyzed by multi-enzyme at a concentration of 0.1 mg / mL on ABTS (%).

[0038] Figure 6 Inhibition rate of walnut peptide hydrolyzed by single-enzyme at a concentration of 0.1 mg / mL on DPPH (%).

[0039] Figure 7 Inhibition rate of walnut peptide hydrolyzed by multi-enzyme at a concentration of 0.1 mg / mL on DPPH (%).

[0040] Figures 2 to 7 In the table, different letters above each column represent significant difference between two groups (p < 0.05), and if the letters are the same, it means that there is no significant difference between the two groups (p > 0.05). For example, if the letters above a certain column are ab, and the letters above other columns are not ab, then there is a significant difference between the two groups. DETAILED DESCRIPTION

[0042] The technical solutions of the present application will be further described below in combination with the description and specific embodiments of the drawings.

[0043] The protein powder used in the examples is wild walnut from Xinjiang, which is broken, ground and defatted to make the protein content reach above 80%. Neutral protease (product catalog number: 9068-59-1), papain (product catalog number: 9001-73-4), pepsin (product catalog number: 9001-75-6), alkaline protease (product catalog number: 9014-01-1), trypsin (product catalog number: 9002-07-7) are all purchased from Shanghai Yuanye Biotechnology Co., Ltd. Flavourzyme (product catalog number: 9001-92-7), complex protease (product catalog number: 9014-01-1) are all purchased from Beijing Suobaolai Technology Co., Ltd.

[0044] Example 1

[0045] This example specifically illustrates the basic steps of preparing the peptide powder in this application, and the main process is: walnut protein powder → dissolution → heating → ultrasonic → enzymolysis → inactivation → centrifugation → ultrafiltration → drying, which is specifically as follows:

[0046] 1) Dissolution: Add water to the walnut protein powder to prepare a mixture with a concentration of about 60 mg / mL, and stir it completely dissolved without clumps under normal temperature conditions with a magnetic stirrer;

[0047] 2) Heating: Heat the completely dissolved mixture in a water bath at 100℃ for about 10 min to further dissolve it;

[0048] 3) ultrasonic: using ultrasonic cell disruptor, the completely dissolved mixture is ultrasonically treated for 10-15 min;

[0049] 4) enzymolysis: adding enzymes into the ultrasonically treated mixture for enzymolysis; adjusting pH to the suitable reaction pH of the enzymes by using HCl or NaOH; the enzymolysis time of each enzyme is 1.5-2.5 h, within which the substrate can be fully enzymolyzed;

[0050] 5) inactivation: placing the enzymolyzed solution in boiling water for 10-15 min for inactivation;

[0051] 6) centrifugation: centrifuging the enzyme-inactivated enzymolyzed solution at a speed of 8000-9000 rpm for 10-15 min, retaining the supernatant and removing the precipitate;

[0052] 7) ultrafiltration: ultrafiltrating the supernatant obtained by enzymolysis using an ultrafiltration cup, and collecting the enzymolyzed solution with a molecular weight of ≤5 kDa;

[0053] 8) drying: drying the collected enzymolyzed solution with a molecular weight of ≤5 kDa by any one of freeze-drying, spray-drying and vacuum-drying, and obtaining walnut protein peptide powder by drying the obtained enzymolyzed solution into powder.

[0054] Example 2

[0055] In this example, the pH and temperature of the applied enzymes are optimized to confirm the optimum conditions of the enzymolysis reaction, which is used for subsequent reactions.

[0056] The enzymes involved in the example include neutral protease, flavor protease, complex protease, papain, pepsin, alkaline protease and trypsin.

[0057] The orthogonal test is used to confirm the enzyme reaction conditions:

[0058] The suitable reaction conditions of neutral protease are pH 6.5-7.5 and temperature 45-55℃;

[0059] The suitable reaction conditions of flavor protease are pH 7.0-8.0 and temperature 45-55℃;

[0060] The suitable reaction conditions of complex protease are pH 7.0-8.0 and temperature 45-55℃;

[0061] The suitable reaction conditions of papain are pH 6.0-7.0 and temperature 55-65℃;

[0062] The suitable reaction conditions of pepsin are pH 2.0-3.0 and temperature 35-40℃;

[0063] The suitable reaction conditions of alkaline protease are pH 9.0-10.0 and temperature 45-55℃;

[0064] The suitable reaction conditions for trypsin are pH 6.5-7.5 and temperature 35-40°C.

[0065] The following experiments are based on the optimal conditions for enzymatic reaction. The enzyme activity of each enzyme added is about 5500-6500 U / g, and the enzyme activity of the enzyme added in the following Examples 3 and 4 is 6000 U / g. The ultrasonic time in the experiment is 10 min, and the drying method is freeze-drying (obtaining freeze-dried powder).

[0066] Example 3

[0067] In this example, walnut powder (3 g) is prepared into walnut peptide powder by single enzyme hydrolysis, including:

[0068] Neutral protease: step 4) adjust the pH value of the mixed solution to 7.0 with 6 M NaOH solution, adjust the temperature to 50°C, add neutral protease 180 mg, and hydrolyze for 2.5 h;

[0069] Flavour protease: step 4) adjust the pH value of the mixed solution to 7.5 with 6 M NaOH solution, adjust the temperature to 50°C, add flavour protease 900 mg, and hydrolyze for 2.5 h;

[0070] Complex protease: step 4) adjust the pH value of the mixed solution to 7.5 with 6 M NaOH solution, adjust the temperature to 50°C, add complex protease 150 mg, and hydrolyze for 2.5 h;

[0071] Papain: step 4) adjust the pH value of the mixed solution to 6.5 with 6 M NaOH solution, adjust the temperature to 60°C, add papain 22.5 mg, and hydrolyze for 2.5 h;

[0072] Pepsin: step 4) adjust the pH value of the mixed solution to 2.5 with 6 M HCl solution, adjust the temperature to 37°C, add pepsin 72 mg, and hydrolyze for 2.5 h;

[0073] Alkaline protease: step 4) adjust the pH value of the mixed solution to 9.0 with 6 M NaOH solution, adjust the temperature to 50°C, add trypsin 90 mg, and hydrolyze for 2.5 h;

[0074] Trypsin: step 4) adjust the pH value of the mixed solution to 7.0 with 6 M NaOH solution, adjust the temperature to 37°C, add trypsin 7.2 mg, and hydrolyze for 2.5 h.

[0075] Example 4

[0076] In this example, walnut powder (3 g) is prepared into walnut peptide powder by single enzyme hydrolysis, including:

[0077] Multi-enzyme walnut No. 1: adjust the pH of the mixed solution to 2.5, adjust the temperature to 37℃, add pepsin 72 mg, and enzymolysis for 2.5 h; adjust the pH of the mixed solution to 7.5, adjust the temperature to 50℃, add flavor protease 900 mg, and enzymolysis for 2.5 h;

[0078] Multi-enzyme walnut No. 2: adjust the pH of the mixed solution to 2.5, adjust the temperature to 37℃, add pepsin 72 mg, and enzymolysis for 2.5 h; adjust the pH of the mixed solution to 6.5, adjust the temperature to 60℃, add papain 22.5 mg, and enzymolysis for 2.5 h.

[0079] Example 5

[0080] This example is directed to the activity evaluation of the walnut protein peptide powder prepared in Examples 3-4, including:

[0081] ① The GABA content of the above-prepared walnut protein peptide powder with sleep-aiding function was determined (OPA method).

[0082] Preparation of reagents: OPA (o-phthaldehyde): weigh 10 mg of OPA powder and dissolve in 1 mL of methanol, avoid light, and store at 4℃ for standby;

[0083] β-mercaptoethanol solution: take 7 μL of β-mercaptoethanol and add to 1 mL of methanol, mix well, and prepare immediately for use;

[0084] Buffer: weigh 50 mg of sodium carbonate and 40 mg of sodium bicarbonate, dissolve in 50 mL of pure water, mix and dissolve, and store at 4℃;

[0085] OPA working solution: take OPA 100 μL, β-mercaptoethanol solution 100 μL, and buffer 800 μL according to the proportion, mix and prepare, and store in the dark after preparation.

[0086] Experimental principle: o-phthaldehyde (OPA) can react with the primary amine group in the sample under alkaline conditions with the participation of β-mercaptoethanol to generate a derivative with strong fluorescence characteristics. The fluorescence intensity or absorbance is proportional to the concentration of γ-aminobutyric acid (GABA). By detecting the signal of the reaction product at a specific wavelength and combining with the standard curve for quantification, the content of GABA in the sample can be accurately determined. As the main inhibitory neurotransmitter of the central nervous system, GABA can reduce nerve excitability and promote sleep and maintain sleep, so this method is not only sensitive and reliable, but also can be used to evaluate the ability of peptide samples to promote GABA production and provide a chemical basis for their sleep-aiding function.

[0087] Detection method: the content of GABA was determined by an enzyme marker instrument, and the operation parameters are shown in Table 1.

[0088] Table 1 Determination of GABA content

[0089] Sample well (μL) OPA (O-Phthaldehyde) 25 β-Mercaptoethanol 25 Sample to be tested 200 Buffer 100

[0090] Specific steps: 100 μL of peptide sample was taken in a 96-well plate. An equal volume of freshly prepared OPA working solution was added to each well, which was prepared by mixing OPA (o-phenylenediamine) 10 mg / mL, dissolved in methanol, stored in the dark, β-mercaptoethanol solution 7 μL / mL, dissolved in methanol, prepared fresh, and sodium carbonate-sodium bicarbonate buffer 50 mg Na2CO3 + 40 mg NaHCO3 dissolved in 50 mL pure water, stored at 4°C. After adding the working solution, the sample and reagent were mixed well, and the derivatization was completed under light-protected conditions for about 15 minutes. After the reaction was completed, the fluorescence intensity (excitation wavelength 340 nm, emission wavelength 455 nm) or absorbance value (about 240 nm) of the derivatized product was measured using a microplate reader, with 3 parallel holes set for each sample. By comparing the GABA standard curve, the GABA content in the sample can be calculated, and the change in GABA level before and after treatment can be compared, thereby indirectly evaluating the ability of the peptide sample to promote GABA production and providing a reliable physicochemical basis for its sleep-promoting function.

[0091] Example 3 Preparation of single-enzyme hydrolyzed walnut protein peptide Figure 1 The GABA content in the sample was calculated according to the GABA standard curve shown, determined by o-phenylenediamine (OPA) derivatization method, and compared with the positive control group (commercial GABA standard, reacted under the same derivatization and detection conditions as the sample group). The results showed that at a concentration of 0.1 mg / mL, the GABA level of the walnut protein peptide treatment group was higher than that of the positive control, suggesting that the peptide may promote the active release of intracellular GABA and has certain sleep-promoting potential. As shown in Figure 2 and Figure 3 As shown, both single-enzyme and multi-enzyme hydrolysis products can increase GABA content to varying degrees, and the GABA production of multi-enzyme hydrolyzed walnut peptide is higher than that of the single-enzyme group. This indicates that multi-enzyme synergistic hydrolysis can optimize the peptide segment structure to some extent and enhance its ability to regulate neurotransmitter levels, thereby playing an active role in sleep-related functions.

[0092] ②ABTS inhibition activity experiment was performed on the above prepared walnut protein peptide powder with sleep-promoting function.

[0093] Preparation of reagents: ABTS free radical stock solution: a mixture of ABTS at a concentration of 7 m M / L and potassium persulfate at a concentration of 2.45 mM / L in phosphate buffer (0.1 M, pH 7.4) was prepared, and the mixture was mixed well and stored at room temperature in the dark for 12-16 h;

[0094] ABTS free radical working solution: the stock solution is generally diluted 40-50 times, so that the absorbance at 734 nm wavelength is 0.70±0.05, which is the ABTS free radical working solution;

[0095] Positive control: 10 mg / mL ascorbic acid (Vc);

[0096] Test sample: weigh an appropriate amount of active peptide powder, and prepare a series of active peptide solutions with distilled water as the solvent.

[0097] Experimental principle: ABTS can be excited to form an oxidant, i.e. ABTS free radical, under the action of persulfate. Antioxidant active substances can interact with ABTS free radicals, thereby reducing the number of ABTS free radicals and making the color of the reaction solution change from dark to light. The absorbance of ABTS free radical at 734 nm wavelength can be measured to evaluate the scavenging ability of antioxidant active substances on ABTS free radical.

[0098] Detection method: ABTS inhibitory activity is determined by enzyme labeling instrument method, and the operation parameters are shown in Table 2.

[0099] Table 2 Determination of ABTS inhibitory activity

[0100] Sample well (μL) Negative control well (μL) Blank well (μL) Sample to be tested 20 20 0 Distilled water 0 0 20 Phosphate buffer solution 0 200 0 ABTS radical working solution 200 0 200

[0101] Specific steps: take 20 μL of sample solution and mix with 200 μL of ABTS free radical working solution, and then react at 37℃ for 30 min. The absorbance value is measured at 734 nm, and an equal volume of ascorbic acid is used as a positive control. The calculation is as follows:

[0102] (1)

[0103] In formula (1), A0 is the value of the blank hole, A1 is the value of the sample hole, and A2 is the value of the negative control hole.

[0104] The ABTS free radical scavenging activity of walnut peptides prepared by single enzyme and multi-enzyme hydrolysis in Examples 3 and 4 is as follows Figure 4 、 Figure 5The results showed that the antioxidant activity of the peptide sample remained stable at a concentration of 0.1 mg / mL. Overall, the antioxidant effect of the multi-enzyme enzyme hydrolysis system was better than that of the single enzyme treatment group. Among them, the combination of pepsin + papain (multi-enzyme walnut No. 2) had the highest scavenging activity, which was better than the combination of pepsin + flavor protease (multi-enzyme walnut No. 1), indicating that reasonable enzyme species matching can more effectively promote the release of antioxidant activity peptide segments. In the single enzyme group, alkaline protease and flavor protease hydrolysate showed strong ABTS free radical scavenging activity, which was the better enzyme species in the single enzyme system. The comprehensive results showed that multi-enzyme synergistic hydrolysis can help to enhance the antioxidant activity of walnut peptide, and this activity is closely related to the function of the central nervous system, suggesting that walnut peptide may promote GABA accumulation by regulating oxidative stress, thereby playing a potential sleep-aiding role.

[0105] ③The prepared walnut protein peptide powder with sleep-aiding function was subjected to DPPH inhibition activity experiment.

[0106] Preparation of reagents: DPPH free radical stock solution: anhydrous methanol as solvent, prepare a DPPH free radical stock solution with a concentration of 1 mM / L;

[0107] DPPH free radical working solution: dilute the stock solution to a concentration of 0.1 mM / L to obtain the working solution;

[0108] Positive control: 10 mg / mL ascorbic acid (Vc);

[0109] Test sample: weigh an appropriate amount of active peptide powder and prepare a series of concentrations of active peptide solution with distilled water as solvent.

[0110] Experimental principle: DPPH is a stable free radical in organic solvents, its alcohol solution is purple, and it needs to be stored at low temperature and away from light, has a single electron, so it can accept an electron or hydrogen ion, and has maximum absorption at a wavelength of 517 nm. When free radical scavengers are present, the single electron of DPPH is captured, causing the color to fade, and the absorbance value at the maximum light absorption wavelength decreases. The decrease in absorbance level indicates an increase in antioxidant activity, which can evaluate the antioxidant capacity of antioxidant active substances.

[0111] Detection method: DPPH inhibition activity was determined by enzyme label instrument method, and the operation parameters are shown in Table 3.

[0112] Table 3 Determination of DPPH inhibition activity

[0113] Sample well (μL) Negative control well (μL) Blank well (μL) Sample to be tested 50 50 0 Distilled water 0 0 50 Anhydrous methanol 0 200 0 DPPH radical working solution 200 0 200

[0114] Specific steps: 50 μL sample solution was mixed with 200 μL DPPH free radical working solution, and the absorbance value was measured at 517 nm after 1 h reaction at 37℃ and 12000 rpm centrifugation for 5 min. Equal volume of ascorbic acid was used as positive control, and the following formula was used to calculate:

[0115] (3)

[0116] In formula (3), A0 is the value of blank hole, A1 is the value of sample hole, and A2 is the value of negative control hole.

[0117] The DPPH free radical scavenging activity of walnut peptides prepared by single enzyme and multi-enzyme hydrolysis in examples 3 and 4 is shown in the following table: Figure 6 、 Figure 7 The results show that in the comparison of enzyme hydrolysis methods, the antioxidant effect of multi-enzyme combination is obviously better than that of single enzyme, and pepsin + flavourzyme is better than pepsin + papain, indicating that the synergistic hydrolysis of enzymes helps to generate high-activity peptide segments. In comparison, the scavenging capacity of single enzyme flavourzyme is relatively low. The results suggest that walnut peptides can maintain the antioxidant homeostasis of cells and promote the maintenance of GABA level, thereby exerting a potential sleep-aiding effect, providing experimental basis for its use as a functional sleep-aiding ingredient.

[0118] IV. Summary of experimental results

[0119] In terms of GABA production promotion, both single enzyme and multi-enzyme hydrolysis products can increase GABA content to different extents, but the GABA production of multi-enzyme hydrolyzed walnut peptides is higher than that of single enzyme group. Among the single enzyme groups, the flavourzyme group, pepsin group and alkaline protease group are better, and the pepsin + flavourzyme combination is the best in the multi-enzyme combination group.

[0120] In terms of free radical scavenging capacity (ABTS scavenging and DPPH scavenging), the scavenging effect of multi-enzyme combination is obviously better than that of single enzyme, and pepsin + papain is more effective in ABTS scavenging, and pepsin + flavourzyme is more effective in DPPH scavenging. For the single enzyme group, the alkaline protease group shows the best comprehensive performance in the two free radical scavenging systems.

[0121] Finally, it should be noted that the above only lists several specific embodiments of the present application. Obviously, the present application is not limited to the above embodiments, and there can be many variations. All variations that can be directly derived or inferred by those skilled in the art from the disclosed content of the present application should be considered as falling within the scope of protection of the present application.

Claims

1. A method for preparing walnut protein peptide powder with sleep-promoting function, characterized in that, The preparation method comprises the following steps: The walnut protein powder is dissolved in water, and the dissolved solution is subjected to ultrasonic treatment, and then enzymes are added for enzymolysis; After the enzymolysis is completed, the added enzymes are inactivated, centrifugation is performed to obtain supernatant, and the supernatant is subjected to ultrafiltration treatment, and then the obtained ultrafiltration liquid is dried into powder, that is, walnut protein peptide powder. The enzymes are selected from one or more of neutral protease, flavor protease, complex protease, papain, pepsin, alkaline protease and trypsin.

2. The production method according to claim 1, wherein The enzymes are selected from a combination of pepsin and papain or a combination of pepsin and flavor protease.

3. The preparation method according to claim 1, characterized in that, Before the neutral protease is added, the pH value of the dissolved solution is adjusted to 6.5-7.5, and the temperature is adjusted to 45-55 DEG C; Before the flavor protease is added, the pH value of the dissolved solution is adjusted to 7.0-8.0, and the temperature is adjusted to 45-55 DEG C; Before the complex protease is added, the pH value of the dissolved solution is adjusted to 7.0-8.0, and the temperature is adjusted to 45-55 DEG C; Before the papain is added, the pH value of the dissolved solution is adjusted to 6.0-7.0, and the temperature is adjusted to 55-65 DEG C; Before the pepsin is added, the pH value of the dissolved solution is adjusted to 2.0-3.0, and the temperature is adjusted to 35-40 DEG C; Before the alkaline protease is added, the pH value of the dissolved solution is adjusted to 8.5-9.5, and the temperature is adjusted to 45-55 DEG C; Before the trypsin is added, the pH value of the dissolved solution is adjusted to 6.5-7.5, and the temperature is adjusted to 35-40 DEG C.

4. The preparation method according to claim 2, characterized in that, Before the pepsin is added, the pH value of the mixed solution is adjusted to 2.0-2.5, the temperature is adjusted to 37-40 DEG C, the pepsin is added, and the enzymolysis is performed for 2.5-3 hours; the pH value of the mixed solution is adjusted to 7.5-8.0, the temperature is adjusted to 50-55 DEG C, the flavor protease is added, and the enzymolysis is performed for 2.5-3 hours; or before the pepsin is added, the pH value of the mixed solution is adjusted to 2.0-2.5, the temperature is adjusted to 37-40 DEG C, the pepsin is added, and the enzymolysis is performed for 2.5-3 hours; the pH value of the mixed solution is adjusted to 6.5-7.0, the temperature is adjusted to 55-60 DEG C, the papain is added, and the enzymolysis is performed for 2.5-3 hours.

5. The production process according to any one of claims 1 to 4, characterized in that, The enzyme activity of the neutral protease, the flavor protease, the complex protease, the papain, the pepsin, the alkaline protease or the trypsin is 5500-6500 U / g.

6. The preparation method according to claim 5, characterized in that, The enzyme activity of the neutral protease, the flavor protease, the complex protease, the papain, the pepsin, the alkaline protease or the trypsin is 6000 U / g.

7. The method of any one of claims 1-6, wherein, After the walnut protein powder is dissolved in water to obtain a mixed solution, the mixed solution is subjected to magnetic stirring at room temperature, and then water bath heating is performed to completely dissolve the walnut protein powder; preferably, the walnut protein powder is prepared by using wild walnuts in Xinjiang, and the walnut protein powder is subjected to crushing, grinding and defatting treatment, so that the protein content is above 80%.

8. The production process according to any one of claims 1 to 7, characterized in that, The ultrafiltration treatment is performed at a molecular weight cut-off of 5 kDa.

9. The walnut protein peptide powder prepared by the preparation method in any one of claims 1-8.

10. The walnut protein peptide powder prepared by the preparation method in any one of claims 1-8 is used for preparing sleep-promoting functional products; the functional products include food and health products.

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