Preparation method of pecan nut protein peptide and beverage thereof

Abstract

The present invention discloses a preparation method of pecan protein peptide and its beverage, belonging to the fields of protein peptide preparation and food. The present invention uses pecans as raw materials, and prepares pecan polypeptide by enzymatic hydrolysis method to obtain the active peptide GAL, which has high antioxidant activity, α-glucosidase and α-amylase inhibitory activities, with a view to developing a pecan protein peptide beverage with delicate and smooth taste, moderate sweetness, and uniform and stable properties, so as to enrich the plant protein beverage products.

Description

Technical Field

[0001] The present invention relates to a preparation method of pecan protein peptide and its beverage, belonging to the fields of protein peptide preparation and food. Background Art

[0002] With the progress of food technology and the change of people's consumption concepts, the research on plant protein beverages has received increasing attention. It is not only rich in plant protein and unsaturated fatty acids, but also does not cause an increase in the cholesterol content in the human body. Plant protein beverages are popular among consumers due to their healthy and natural ingredients and unique delicate taste.

[0003] Food protein-derived active peptides have rich nutritional value and various physiological functions such as antioxidant, anti-fatigue, and immune regulation, and are widely used in foods. In the future, it is expected to replace nutrients such as proteins and amino acids. Wang Duan used kudzu root and walnut kernels as the main raw materials, adopted the neutral protease hydrolysis method, and combined it with lactic acid bacteria to make a new type of nut polypeptide beverage. Guo Xianhe used papain and alkaline protease to hydrolyze hazelnut protein to prepare a hazelnut polypeptide solid beverage. Yang Xu et al. enzymatically hydrolyzed black kidney beans with flavor protease, added 15% white granulated sugar and 0.03% citric acid to make a black kidney bean peptide beverage with a better taste. Yang Bo et al. extracted peanut protein from defatted peanut meal powder by the alkali solution acid precipitation method and hydrolyzed it with subtilisin to finally make a delicious beverage.

[0004] Patent CN116671557A discloses the use of composite cellulose and protease to enzymatically hydrolyze pecans to prepare pecan protein beverages; patent CN117413885A discloses the preparation of pecan polypeptides that can be used to activate the activity of ADH enzyme; the paper "Preparation, Screening, Identification and Application Research of Pecan Protein and Hypolipidemic Peptides" discloses that the prepared pecan polypeptides can inhibit α-glucosidase and pancreatic lipase. The development of the functions of pecan protein peptides in the above technical solutions is still relatively limited.

[0005] Therefore, developing a multi-functional pecan active peptide has high edible value and economic value, and further expands the application of pecans. Summary of the Invention

[0006] To solve the above problems, the present invention uses pecans as raw materials, prepares pecan polypeptides by enzymatic hydrolysis method, and further screens out short peptide GAL (Gly-Ala-Leu) with strong antioxidant activity and different intermediate product active peptides H2 and L3, in order to develop a pecan protein peptide beverage with delicate and smooth taste, moderate sweetness, and uniform and stable properties, and enrich the products of plant protein beverages.

[0007] The first object of the present invention is to provide an active peptide, and the amino acid sequence of the peptide is GAL (Gly-Ala-Leu).

[0008] The present invention also provides a nucleic acid molecule for encoding the above-mentioned active peptide.

[0009] The present invention also provides a biological material containing the above nucleic acid molecule, and the biological material is an expression cassette, a transposon, a plasmid vector, a viral vector, an engineered bacterium or a transgenic cell line.

[0010] The second object of the present invention is to provide a preparation method of the above-mentioned active peptide, characterized in that the method includes preparation through pecan, synthesis through genetically engineered bacteria or artificial synthesis.

[0011] In one embodiment, the method for preparation through pecan is as follows:

[0012] (1) After shelling the pecan, soak it in sodium hydroxide solution, peel, dry and crush to obtain pecan powder;

[0013] (2) Mix the pecan powder with water, adjust the pH to 10-12, perform water bath and centrifugation to obtain the supernatant, adjust the pH of the supernatant to 4-5, and centrifuge to obtain the precipitate, namely pecan protein;

[0014] (3) Use papain to enzymatically hydrolyze the pecan protein to prepare pecan polypeptide;

[0015] (4) Use Sephadex G-15 dextran gel column to separate the pecan polypeptide, and collect the active peptide H2 at the ultraviolet absorption peak of 280 nm;

[0016] (5) Perform purification by preparative liquid phase, use a reverse phase column to separate the active peptide H2, and prepare the active peptide L3, and the proportion of GAL in L3 reaches 90.73%.

[0017] In one embodiment, the method for preparation through pecan is as follows:

[0018] (1) After shelling the pecan, soak it in a sodium hydroxide solution with a concentration of 0.2-0.6% w / v, heat to 85-95 °C and stir for 10-20 min, peel, dry at 55-65 °C for 6-10 h, and crush to obtain pecan powder;

[0019] (2) Mix the pecan powder with water according to the dosage ratio of 1 g: 15-30 mL, adjust the pH to 10-12, perform a water bath at 50-80 °C for 1-2 h, ultrasonic treatment for 5-30 min, centrifuge to obtain the supernatant, adjust the pH of the supernatant to 4-5, and centrifuge to obtain the precipitate, namely pecan protein;

[0020] (3) Use papain (addition amount 500 - 10000 U / g) to enzymatically hydrolyze pecan protein (substrate concentration 1040 g / L), and carry out enzymatic hydrolysis at pH 5.5 - 7.0 and 30 - 55 °C for 1 - 4 h to prepare pecan polypeptide;

[0021] (4) Use Sephadex G - 15 dextran gel column to separate pecan polypeptide. When the ultraviolet absorption peak is at 280 nm and the elution time is 43.6 min, collect the active peptide H2;

[0022] (5) Based on preparative liquid chromatography, use a reverse - phase column to separate the active peptide H2. After multiple purifications at an ultraviolet absorption peak of 280 nm, prepare the active peptide L3, and the proportion of GAL in L3 reaches 90.73%.

[0023] The third object of the present invention is to provide the application of the above - mentioned active peptide, or the above - mentioned nucleic acid molecule, or the above - mentioned biological material in the preparation of antioxidant products or products for inhibiting the activities of α - glucosidase and α - amylase, and the products include pharmaceuticals, health products or cosmetics.

[0024] The fourth object of the present invention is to provide the application of pecan polypeptide, or active peptide H2, or active peptide L3 in the preparation of antioxidant products or products for inhibiting the activities of α - glucosidase and α - amylase, and the products include pharmaceuticals, health products or cosmetics;

[0025] The preparation method of the pecan polypeptide is as follows:

[0026] (1) After shelling pecans, soak them in sodium hydroxide solution, peel, dry, and crush to obtain pecan powder;

[0027] (2) Mix the pecan powder with water, adjust the pH to 10 - 12, carry out water bath and centrifugation to obtain the supernatant, adjust the pH of the supernatant to 4 - 5, and centrifuge to obtain the precipitate, which is pecan protein;

[0028] (3) Use papain to enzymatically hydrolyze pecan protein to prepare pecan polypeptide;

[0029] The preparation method of the active peptide H2 is as follows:

[0030] Use Sephadex G - 15 dextran gel column to separate pecan polypeptide, and collect the active peptide H2 at an ultraviolet absorption peak of 280 nm;

[0031] The preparation method of the active peptide L3 is as follows:

[0032] Use a reverse - phase column of preparative liquid chromatography to separate the active peptide H2 to prepare the active peptide L3.

[0033] In one embodiment, when preparative liquid chromatography is used and a reverse-phase column is employed to separate active peptide H2, after multiple purifications, at a UV absorption peak of 280 nm and an elution time of 6.3 min, active peptide L3 is collected.

[0034] The fifth object of the present invention is to provide a product containing one or more of pecan polypeptide, active peptide H2, active peptide L3, and active peptide GAL.

[0035] In one embodiment, the preparation method of the pecan polypeptide is as follows:

[0036] (1) After shelling the pecans, they are soaked in a sodium hydroxide solution, peeled, dried, and pulverized to obtain pecan powder.

[0037] (2) The pecan powder is mixed with water, the pH is adjusted to 10 - 12, and after water bath and centrifugation, the supernatant is obtained. The pH of the supernatant is adjusted to 4 - 5, and centrifugation gives a precipitate, which is pecan protein.

[0038] (3) The pecan protein is enzymatically hydrolyzed using papain to prepare pecan polypeptide.

[0039] The preparation method of the active peptide H2 is as follows:

[0040] The pecan polypeptide is separated using a Sephadex G-15 dextran gel column. At a UV absorption peak of 280 nm and an elution time of 43.6 min, active peptide H2 is collected.

[0041] The preparation method of the active peptide L3 is

[0042] Using a preparative liquid chromatograph, active peptide H2 is separated through a reverse-phase column. At a UV absorption peak of 280 nm and an elution time of 6.3 min, active peptide L3 is prepared.

[0043] The preparation method of the active peptide GAL is as follows:

[0044] The method includes preparation from pecans, synthesis by genetically engineered bacteria, or artificial synthesis.

[0045] The sixth object of the present invention is to provide a pecan protein beverage containing pecan protein peptide, and the preparation method of the beverage is as follows:

[0046] Raw material composition (w / v; g / 100 mL): 1 - 3% of pecan protein peptide, 1 - 24% of milk powder, 0.5 - 3% of granulated sugar, 0.1 - 0.3% of stabilizer monoglyceride, 0 - 0.05% of sodium carboxymethylcellulose, 0 - 0.08% of xanthan gum, and water are mixed evenly and sheared for 1 - 5 min; sterilized at 60 - 83 °C for 20 - 40 min to prepare.

[0047] The pecan protein peptide includes one or more of pecan polypeptide, active peptide H2, active peptide L3, and active peptide GAL.

[0048] Advantages of the present invention

[0049] The present invention uses pecan as raw material, prepares pecan polypeptide by enzymatic hydrolysis method, and further screens to obtain short peptide GAL (Gly-Ala-Leu) with strong antioxidant activity and different intermediate product active peptides H2 and L3, aiming to develop a pecan protein peptide beverage with delicate and smooth taste, moderate sweetness, and uniform and stable properties, and enrich the plant protein beverage products;

[0050] Specifically:

[0051] 1. When the concentration of the active peptide GAL (Gly-Ala-Leu) prepared by the present invention is 0.1 mg / mL, the DPPH scavenging rate reaches 86.1%;

[0052] 2. The pecan polypeptide, active peptide H2, active peptide L3, and active peptide GAL prepared by the present invention have excellent α-glucosidase and α-amylase inhibitory activities. Among them, the inhibitory activity of active peptide GAL is the strongest, and the IC 50 values for α-glucosidase and α-amylase are 0.14 mg / mL and 0.25 mg / mL respectively;

[0053] 3. The pecan protein beverage prepared based on pecan polypeptide, active peptide H2, active peptide L3, and active peptide GAL by the present invention has strong stability and good sensory flavor, providing technical support for the high-value utilization of pecan. Description of the drawings

[0054] Figure 1 For the total reducing power detection of active peptide H2;

[0055] Figure 2 For the DPPH scavenging ability detection of active peptide H2. Detailed implementation manners

[0056] The following are the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0057] Raw materials:

[0058] The pecans are purchased from: Tangchen Agricultural Development Co., Ltd., Ma'anshan, Anhui;

[0059] Papain is purchased from: Aladdin Reagent Co., Ltd.

[0060] Experimental methods

[0061] 1. RP-HPLC purification method:

[0062] Weigh a certain amount of the sample, dissolve it with an appropriate amount of ultrapure water to prepare a sample solution of the above-screened components with a concentration of 20 mg / mL, and purify it by preparative high-performance liquid chromatography. Inject the sample solution into a reverse-phase column (Waters, C18 chromatographic column), and the mobile phase is methanol at 75% (v / v) containing 0.08% (v / v) trifluoroacetic acid (TFA). Collect the elution fractions according to the ultraviolet absorption peak, with the ultraviolet absorption wavelength at 280 nm, and at the same time collect the components according to the mass spectrometry peak, and the flow rate is 0.5 mL / min.

[0063] 2. Determination of total reducing ability

[0064] Refer to the general method. Take 1.0 mL of sample solutions with concentrations of 0.01, 0.05, 0.1, 0.5, 1, 3, 5, 7, and 9 mg / mL respectively. Add 1.0 mL of phosphate buffer with a pH of 6.6 to each concentration of the sample solution, mix well, and then add 1.0 mL of 1% potassium ferricyanide solution. React in a water bath at 50 °C for 20 min, and then add 1.0 mL of 10% trichloroacetic acid solution at room temperature, and centrifuge at a speed of 4000 r / min for 10 min. Mix 1.0 mL of the supernatant with 1.0 mL of ferric chloride solution. Measure the absorbance at a wavelength of 700 nm. The higher the absorbance value, the better the total reducing ability of the sample solution.

[0065] 3. DPPH scavenging ability

[0066] Refer to the general method. Accurately weigh 5 mg of DPPH powder and mix it with 95% ethanol and make up the volume to 100 mL to obtain a DPPH treatment solution with a concentration of 50.0 μg / mL. Take 2.0 mL of the sample solutions with the above concentrations respectively, and at the same time take 2.0 mL of the DPPH treatment solution and mix them well, and incubate in the dark at room temperature for 30 min. The blank control group is 95% ethanol solution, and measure the absorbance at a wavelength of 517 nm. Take the average value of the results of three parallel experiments and calculate the scavenging rate of different concentration sample solutions.

[0067] 4. α-Amylase inhibitory activity

[0068] α - amylase hydrolyzes starch into reducing sugars. 3,5 - dinitrosalicylic acid reacts with reducing sugars to show a reddish - brown color. Colorimetric determination is carried out at 540 nm to quantitatively measure the activity of α - amylase. Referring to the DNS (3,5 - dinitrosalicylic acid) colorimetric method, the reaction system: After incubating 0.3 mL of α - amylase (1 U / mL) and a certain concentration of inhibitor solution in a 37 °C water bath for 5 min, add 0.4 mL of starch solution (1%) to initiate the reaction. After the system reacts at 37 °C for 5 min, add 0.2 mL of DNS, react in a boiling water bath for 5 min, take it out and cool to a constant volume of 10 mL, and finally measure the absorbance value at 540 nm. The enzyme inhibition test is repeated 3 times, with 3 replicates each time.

[0069] 5. Inhibitory activity of α - glucosidase

[0070] Principle: Detect that p - nitrophenyl - β - D - galactoside (PNPG) reacts with α - glucosidase to generate yellow - green p - nitrophenol (PNP), and judge the inhibitory activity of the sample according to the absorbance of the solution.

[0071] Add 100 μL of PNPG (10 mmol / L) to 850 μL of phosphate buffer (PH = 6.8). Then add the sample solution, and use a blank control (reaction system without enzyme) to eliminate the influence of p - nitrophenol (PNP) generated by the oxidation of PNPG itself on the measurement result. Incubate at 37 °C for 10 min, add 50 μL of enzyme (2 U / mL), incubate for another 50 min, and add 1 mL of Na2CO3 to terminate the reaction. Measure the absorbance at 405 nm.

[0072] Example 1: Preparation of pecan active peptides

[0073] 1. Preparation of pecan powder

[0074] Shell the pecan kernels, soak the kernels in a NaOH solution with a concentration of 0.4% w / v, heat to 90 °C and stir for 10 min, remove the skin, and rinse thoroughly; dry in a constant - temperature drying oven at 60 °C for 8 h, and then pulverize with a blender to obtain pecan powder.

[0075] 2. Preparation of pecan protein

[0076] Mix the pecan powder and distilled water in a ratio of 1:20 (g:mL), adjust the pH value to 11 with sodium bicarbonate solution, and keep it in a water bath at 55 °C for 1 h; centrifuge at 4500 rpm for 20 min to obtain the supernatant, adjust the pH value of the supernatant to 4.5 with citric acid solution (concentration of 30% w / v), and centrifuge at 5000 rpm for 10 min. The precipitate is pecan protein.

[0077] 3. Preparation of pecan polypeptides

[0078] Take the pecan protein prepared in step 2, mix it with water to prepare a pecan protein solution with a concentration of 30 mg / mL, heat it in a water bath to 70 °C, ultrasonically treat it for 30 min, add papain (5000 U / g), and enzymatically hydrolyze it at pH 6.5 and 50 °C for 4 h to prepare pecan polypeptides.

[0079] 4. Preparation of hypoglycemic potential active peptides

[0080] Further separate the pecan protein hydrolysate using a Sephadex G-15 gel column. Separation is carried out through a Sephadex G-15 gel filtration column equilibrated with Tris-acetate buffer (pH 7.0), and the column is eluted with ultrapure water at a flow rate of 0.5 mL / min.

[0081] Collect the elution fractions according to the ultraviolet absorption peak (280 nm) to obtain different peak components, which are respectively named H1 (concentration: 5.6 mg / mL; peak elution time: 21.3 min), H2 (concentration: 18.9 mg / mL; peak elution time: 43.6 min), and H3 (concentration: 13.6 mg / mL; peak elution time: 71.5 min). Measure the α-glucosidase inhibitory activity and α-amylase inhibitory activity of the three components. Among them, H2 shows good inhibitory abilities against α-amylase and α-glucosidase, and its IC50 values are 0.46 mg / mL and 0.58 mg / mL respectively.

[0082] 5. Preparation of active peptides

[0083] Take the active peptide H2 prepared in step 4, prepare a 20 mg / mL solution, and purify it by preparative liquid chromatography. Inject the sample solution into a reverse-phase column (waters, C18 chromatographic column), separate and purify different peak components according to the ultraviolet absorption peak (280 nm), detect its α-glucosidase inhibitory activity and α-amylase inhibitory activity. After multiple purifications, obtain the active peptide L3 (content reaches 97.5 g / 100 g; peak elution time: 6.3 min), and its IC values for α-glucosidase and α-amylase 50 are 0.17 mg / mL and 0.26 mg / mL respectively.

[0084] Use LC-ESI-Orbitrap MS / MS method to identify the amino acid sequence of the active peptide L3. The mass spectrometry identification results of the L3 component show that the main peptide sequence in L3 is GAL, and its abundance ratio in the sample is 90.73%.

[0085] 6. Preparation of GAL peptide

[0086] Entrust a biological company to complete the synthesis of the GAL peptide. The purity of the synthesized peptide segment is identified by liquid chromatography-mass spectrometry to be above 95%.

[0087] Example 2: Detection of Polypeptide Performance

[0088] Take the pecan polypeptide, active peptide H2, active peptide L3, and GAL peptide prepared in Example 1 to detect the polypeptide performance.

[0089] 1. Detection of Antioxidant Capacity

[0090] The antioxidant components in the pecan polypeptide can effectively reduce potassium ferricyanide. The higher the absorbance value, the better the reduction performance of the sample.

[0091] The total reducing power detection results of active peptide H2 are as Figure 1 shown. The results show that with the increase of concentration, the total reducing ability of active peptide H2 increases accordingly. When the concentration is 9 mg / mL, its absorbance value is 0.658, reaching 36.41% of VC, indicating that the pecan polypeptide has a certain reducing ability.

[0092] The DPPH scavenging results of H2 are as Figure 2 shown. The results show that when the concentration is 9 mg / mL, the highest DPPH scavenging ability of pecan polypeptide H2 is 86.25%, and the antioxidant property is the strongest.

[0093] Detect the antioxidant capacity of L3 and active peptide GAL in the same way. The results show that when the active peptide GAL (Gly-Ala-Leu) is at a concentration of 0.1 mg / mL, the DPPH scavenging rate reaches 86.1%; when L3 is at a concentration of 0.1 mg / mL, the DPPH scavenging rate reaches 77.2%.

[0094] 2. α-Glucosidase and α-Amylase Inhibitory Activity

[0095] The half-inhibitory concentration results of α-amylase of pecan polypeptide, H2, L3, and GAL are shown in Table 1. The results show that the synthesized GAL has the strongest inhibitory effect, followed by L3 and H2, and the pecan polypeptide has the weakest inhibitory effect.

[0096] Table 1 Inhibitory Activity of α-Amylase

[0097]

[0098] The half-inhibitory concentration results of α-glucosidase of pecan polypeptide, H2, L3, and GAL are shown in Table 2. The results show that the synthesized GAL has the strongest inhibitory effect, followed by L3 and H2, and the pecan polypeptide has the weakest inhibitory effect.

[0099]

[0100] Although both α-glucosidase and α-amylase belong to the glycoside hydrolase family, there are significant differences in their functions and active sites. α-Amylase and its function: α-Amylase, also known as endo-amylase, can randomly hydrolyze the α-1,4-glycosidic bonds inside starch. When the substrate is amylose, the hydrolysis products are glucose, maltose, and maltotriose; when the substrate is amylopectin, the hydrolysis products are glucose, maltose, maltotriose, and α-dextrin containing more than 3 glucose residues with α-1,6-glycosidic bonds.

[0101] Glucosidase is a large class of enzymes in the glycoside hydrolase family, and its main function is to hydrolyze glucosidic bonds to release glucose as a product. That is, α-glucosidase and α-amylase are two enzymes with completely different functions and actions.

[0102] Example 3: Preparation of pecan peptide protein beverage

[0103] To prepare the pecan peptide protein beverage, the technological process is as follows:

[0104] Pecan kernels → impurity removal → soaking → peeling → crushing → protein → pecan active peptide → optimization of peptide-milk ratio → sweetening with granulated sugar → optimization of stabilizer ratio → shearing and homogenization → filling → sterilization → finished product.

[0105] The specific steps are as follows:

[0106] Raw material composition (w / v; g / 100 mL): 3% pecan polypeptide, 15% milk powder, 3% granulated sugar, 0.1% monoglyceride as stabilizer, 0.05% sodium carboxymethylcellulose, 0.02% xanthan gum, and water; after mixing the above raw materials evenly, stir at 1000 r / min for 10 min; sterilize at 65 °C for 30 min to prepare the pecan peptide protein beverage.

[0107] Example 4: Preparation of pecan peptide protein beverage

[0108] On the basis of Example 3, change the pecan polypeptide to active peptide H2, and the remaining steps are the same as those in Example 1 to prepare the pecan peptide protein beverage.

[0109] Example 5: Preparation of pecan peptide protein beverage

[0110] On the basis of Example 3, change the pecan polypeptide to active peptide L3, and the remaining steps are the same as those in Example 1 to prepare the pecan peptide protein beverage.

[0111] Example 6: Preparation of pecan peptide protein beverage

[0112] On the basis of Example 3, change the pecan polypeptide to active peptide GAL, and the remaining steps are the same as those in Example 1 to prepare the pecan peptide protein beverage.

[0113] Take the pecan peptide protein beverages prepared in Examples 3 to 6 and conduct sensory evaluation.

[0114] The results show that the pecan peptide protein beverages prepared in Examples 3 to 6 have good sensory characteristics. After being sterilized at 100 °C for 30 minutes, the state is stable, the beverage is clear, and the pecan protein peptide beverage remains stable and homogeneous during the 3-month storage period.

[0115] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. Use of active peptide GAL in the preparation of a drug having both antioxidant and hypoglycemic functions, characterized in that, The blood sugar lowering is achieved by inhibiting the activities of α-glucosidase and α-amylase.

2. Use of active peptide GAL in the preparation of a health product having both antioxidant and hypoglycemic adjuvant functions, characterized in that, The assisting in blood sugar lowering is achieved by inhibiting the activities of α-glucosidase and α-amylase.

3. The application according to any one of claims 1 to 2, characterized in that The active peptide GAL is prepared from pecan nuts or synthesized by genetically engineered bacteria or artificially synthesized.

4. The application according to claim 3, characterized in that, The method for preparing from pecan nuts is as follows: (1) After shelling the pecan nuts, soak them in sodium hydroxide solution, peel, dry, and crush to obtain pecan nut powder; (2) Mix the pecan nut powder with water, adjust the pH to 10 - 12, perform water bath and centrifugation to obtain the supernatant, adjust the pH of the supernatant to 4 - 5, and centrifuge to obtain the precipitate, which is pecan nut protein; (3) Use papain to enzymatically hydrolyze the pecan nut protein to prepare pecan nut polypeptide; (4) Use Sephadex G-15 dextran gel column to separate the pecan nut polypeptide, and collect the active peptide H2 at a UV absorption peak of 280 nm; (5) Use a reverse-phase column to screen the active peptide H2 to prepare the active peptide L3, and the proportion of GAL in L3 reaches 90.73%.

Images