Method for preparing wheat germ albumin anti-fatigue peptide through enzymatic hydrolysis

Abstract

The invention discloses a method for preparing a wheat germ protein anti-fatigue peptide by an enzyme method. Taking a certain amount of defatted wheat germ powder, mixing the defatted wheat germ powder with deionized water according to a material-liquid ratio of 1: 10 (W / V), shaking the mixture on a shaking table at 30 DEG C for 1.5 h, centrifuging the extract at 4 DEG C at 8000 r / min for 20 min, taking the supernatant, and freeze-drying the supernatant; the method comprises the following steps: optimizing enzymolysis conditions, finding out optimal enzymolysis conditions, stirring and adjusting the pH value of a mixed solution to be 6, adding papain according to the enzyme addition amount of 8000U / g and the substrate concentration of 2%, putting into a water bath kettle at 55 DEG C, reacting for 2 hours, immediately raising the temperature of an enzymolysis system to 100 DEG C after the reaction is finished, deactivating enzyme for 10 minutes, and stopping the reaction, so as to obtain an enzymolysis peptide solution with oxidation resistance; and the enzymolysis peptide liquid is subjected to ultrafiltration to obtain peptide fragments in five molecular weight intervals, and the peptide fragment with the optimal oxidation resistance is found, so that effective utilization of high-quality protein product resources is realized.

Description

Technical Field

[0001] The invention belongs to the field of food additives and functional food ingredients, and specifically is a method for preparing wheat germ albumin anti-fatigue peptides by enzymatic hydrolysis. Background Art

[0002] Due to the toxicity and side effects of synthetic antioxidants, the use of agricultural product processing byproducts to prepare natural antioxidants has attracted widespread attention from scholars and has become a hot topic in the fields of food, medicine and cosmetics at home and abroad. Studies have found that wheat germ protein contains many biologically active amino acid sequences, which can release bioactive peptides such as antihypertensive peptides, anti-fatigue peptides and antibacterial peptides under the action of proteases, which provides new ideas and approaches for the in-depth development of wheat germ.

[0003] At present, the methods generally used to prepare wheat germ bioactive peptides include artificial synthesis, chemical hydrolysis, enzymatic hydrolysis, direct extraction and microbial fermentation. The peptide bonds of proteins are broken under the action of proteases, but the structure and configuration of amino acids remain unchanged. No toxic and harmful substances are produced during the enzymatic hydrolysis process. It has the advantages of high safety, mild hydrolysis conditions, easy control of the hydrolysis process, simple equipment, and low production cost. It has now been widely recognized. Moreover, after appropriate enzymatic hydrolysis, the functional properties of proteins can be improved to a certain extent, such as improved solubility and emulsification, reduced allergenicity, easy digestion and absorption by the human body, and improved biological activity of proteins. Wheat germ protein contains a variety of amino acid sequences with different biological activities, and different proteases can be used to hydrolyze biologically active peptides. At the same time, wheat germ active peptides also have certain acid resistance and good thermal stability, water solubility and other characteristics, so that they can be added to various foods as functional factors. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing wheat germ albumin anti-fatigue peptides by enzymatic hydrolysis, which provides a reference for the development of related products and realizes the effective utilization of high-quality protein by-product resources. The method takes the free radical scavenging effect of the enzymatic hydrolysis system as an indicator, optimizes the enzymatic hydrolysis reaction conditions, and then ultrafilters to obtain wheat germ albumin anti-fatigue peptides with obvious antioxidant properties. The process of the present invention is simple to operate, has low equipment and manpower investment, good repeatability, and reduces the waste and pollution of water resources.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0006] A method for preparing anti-fatigue peptides from wheat germ albumin by enzymatic hydrolysis, characterized in that: the method controls the enzymatic hydrolysis reaction conditions and ultrafilters the enzymatic peptide solution to obtain peptide segments with good free radical scavenging effect, and the specific steps are as follows:

[0007] (1) Take a certain amount of defatted wheat germ powder, mix it with deionized water at a solid-liquid ratio of 1:10 (W / V), shake it on a shaker at 30°C for 1.5 h, centrifuge the extract at 8000 r / min for 20 min at 4°C, take the supernatant and freeze-dry it;

[0008] (2) adjusting the pH value of the mixed solution to 6 by stirring, adding papain at an enzyme amount of 8000 U / g and a substrate concentration of 2%;

[0009] (3) placing the mixture in a water bath at 55°C for 2 h, and immediately raising the temperature of the enzymatic hydrolysis system to 100°C for 10 min to inactivate the enzyme and obtain an enriched reaction solution containing anti-fatigue peptides.

[0010] (4) The enzymatic peptide solution was passed through ultrafiltration membranes of 10KDa, 5KDa, 3KDa and 1KDa to obtain five peptide fragments: MW>10kDa, MW5–10kDa, MW3–5kDa, MW1–3kDa and MW<1kDa;

[0011] (5) The free radical scavenging activity of the five peptides was measured to find the peptide with better antioxidant properties.

[0012] As an improvement, the protein source of the peptide is wheat germ byproduct after defatting wheat germ.

[0013] The advantages of the present invention compared with the prior art are:

[0014] The anti-fatigue peptide prepared by using defatted wheat germ as raw material has low production cost, simple process operation, small equipment and manpower investment, good repeatability, reduced waste and pollution of water resources, and significantly improved the antioxidant performance of the enzymatic hydrolysis product. The anti-fatigue peptide prepared by the present invention not only has a relatively significant effect of scavenging free radicals but also is safe and nutritious, provides a reliable base material or ingredient for the development of natural preservatives and functional antioxidant foods, can improve the quality and safety of my country's antioxidant products, and meet the requirements of the development of the natural food additive market. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a line graph of DPPH free radical scavenging rate under different substrate concentrations of the present invention;

[0016] Figure 2 It is a line graph of DPPH free radical scavenging rate under different pH conditions of the present invention;

[0017] Figure 3 It is a line graph of DPPH free radical scavenging rate under different temperatures of the present invention;

[0018] Figure 4 It is a line graph of DPPH free radical scavenging rate under different enzyme amounts of the present invention;

[0019] Figure 5 The structural diagrams of five peptide segments of the present invention;

[0020] Figure 6 It is a bar graph of the DPPH free radical scavenging rates of F1, F2, F3, F4, F5, wheat germ albumin, and VC of the present invention;

[0021] Figure 7 The bar graph of the ABTS free radical scavenging rate of five peptides using VC as a positive control in the present invention;

[0022] Figure 8 The bar graph of hydroxyl radical scavenging rate using VC as a positive control in the present invention;

[0023] Fig. 9 This is a bar graph of Fe2+ chelating ability using VC as a positive control in the present invention. DETAILED DESCRIPTION

[0024] The specific implementation of the present invention will be further described below in conjunction with the accompanying drawings.

[0025] In order to make the contents of the present invention more clearly understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0026] Example 1

[0027] Mix 30g defatted wheat germ powder and 300mL water, shake on a shaker at 30℃ for 1.5h, centrifuge the extract at 8000r / min for 20min at 4℃, take the supernatant and freeze-dry. Adjust the substrate concentration to 1%, 2%, 3%, 4%, and 5%, add 8000U / g of papain, place the resulting enzymatic hydrolysis system solution at 50℃ for 2h, and immediately raise the temperature of the enzymatic hydrolysis system to 100℃ to inactivate the enzyme for 10min to terminate the reaction, and obtain an enzymatic hydrolysis peptide solution with antioxidant properties. Peptides in five molecular weight ranges were obtained, and freeze-dried powdered anti-fatigue peptides were prepared, such as Figure 1 As shown, the hydrolysis degrees of 1%, 2%, 3%, 4% and 5% substrate concentrations were 2.6%, 4.6%, 2.7%, 2.4% and 2.2%, respectively, and the DPPH free radical scavenging rates were 52%, 62.9%, 55.8%, 45% and 44.8%, respectively.

[0028] Implementation Example 2

[0029] 30g defatted wheat germ powder and 300mL water were mixed and shaken on a shaker at 30℃ for 1.5h. The extract was centrifuged at 8000r / min for 20min at 4℃. The supernatant was taken and freeze-dried. The substrate concentration was adjusted to 2%, the pH was adjusted to 5.0, 5.5, 6.0, 6.5, and 7.0 respectively, 8000U / g of papain was added, and the resulting enzymatic hydrolysis system solution was placed at 50℃ for 2h. After the reaction, the temperature of the enzymatic hydrolysis system was immediately raised to 100℃ to inactivate the enzyme for 10min to terminate the reaction, and an enzymatic hydrolysis peptide solution with antioxidant properties was obtained. Peptides in five molecular weight ranges were obtained, and the powdered anti-fatigue peptide segments were prepared by freeze drying, such as Figure 2 As shown, the hydrolysis degrees at pH 5.0, 5.5, 6.0, 6.5 and 7.0 were 2.6%, 2.8%, 5.4%, 4.4% and 4.2%, respectively, and the DPPH free radical scavenging rates were 65.3%, 65.1%, 66.6%, 61% and 53.4%, respectively.

[0030] Implementation Example 3

[0031] 30g defatted wheat germ powder and 300mL water were mixed and shaken on a shaker at 30℃ for 1.5h. The extract was centrifuged at 8000r / min for 20min at 4℃. The supernatant was taken and freeze-dried. The substrate concentration was adjusted to 2%, the amount of papain added was adjusted to 4000U / g, 6000U / g, 8000U / g, 10000U / g, 12000U / g, respectively, the pH was adjusted to 5.0, and the resulting enzymatic hydrolysis system solution was placed at 50℃ for 2h. After the reaction, the temperature of the enzymatic hydrolysis system was immediately raised to 100℃ for 10min to inactivate the enzyme and terminate the reaction. The enzymatic hydrolysis peptide solution with antioxidant properties was obtained, and peptides in five molecular weight ranges were obtained. The powdered anti-fatigue peptide segments prepared by freeze drying were as follows: Figure 3 As shown, the hydrolysis degrees of 4000U / g, 6000U / g, 8000U / g, 10000U / g and 12000U / g were 1.6%, 1.7%, 4.2%, 2.5% and 2% respectively, and the DPPH free radical scavenging rates were 54.6%, 59.1%, 62.9%, 60% and 53.7% respectively.

[0032] Implementation Example 4

[0033] 30g defatted wheat germ powder and 300mL water were mixed and shaken on a shaker at 30℃ for 1.5h. The extract was centrifuged at 8000r / min for 20min at 4℃. The supernatant was taken and freeze-dried. The substrate concentration was adjusted to 2%, the amount of papain added was adjusted to 8000U / g, the pH was adjusted to 5.0, and the resulting enzymatic hydrolysis system solution was placed at 45℃, 50℃, 55℃, 60℃, and 65℃ for 2h. After the reaction, the temperature of the enzymatic hydrolysis system was immediately raised to 100℃ to inactivate the enzyme for 10min to terminate the reaction, and an enzymatic hydrolysis peptide solution with antioxidant properties was obtained. The powdered anti-fatigue peptide was prepared by freeze-drying, such as Figure 4 As shown, the hydrolysis degrees at temperatures of 45°C, 50°C, 55°C, 60°C and 65°C were 1.6%, 2.5%, 4.2%, 2.6% and 3.2%, respectively, and the DPPH free radical scavenging rates were 37.3%, 47.9%, 62.9%, 52% and 59.7%, respectively.

[0034] Implementation Example 5

[0035] The enzymatic peptide solution was passed through 10KDa, 5KDa, 3KDa, and 1KDa ultrafiltration membranes to obtain five peptide segments (such as F1 (MW>10kDa), F2 (MW5–10kDa), F3 (MW 3–5kDa), F4 (MW 1–3kDa), and F5 (MW<1kDa). Figure 5 ).

[0036] Implementation Example 6

[0037] Mix 30g defatted wheat germ powder and 300mL water, shake on a shaker at 30℃ for 1.5h, centrifuge the extract at 8000r / min for 20min at 4℃, take the supernatant and freeze-dry. Adjust the substrate concentration to 2%, the amount of papain added to 8000U / g, and the pH to 5.0. Place the resulting enzymatic hydrolysis system solution at 45℃, 50℃, 55℃, 60℃, and 65℃ for 2h. Immediately raise the temperature of the enzymatic hydrolysis system to 100℃ to inactivate the enzyme for 10min to terminate the reaction, and obtain an enzymatic hydrolysis peptide solution with antioxidant properties. The powdered anti-fatigue peptide was freeze-dried to determine the DPPH free radical scavenging activity of the five peptide segments and wheat germ albumin, and VC was used as a positive control. Figure 6 As shown, the DPPH free radical scavenging rates of F1, F2, F3, F4, F5, wheat germ albumin and VC were 36.63%, 27.4%, 40.33%, 34.53%, 35.83%, 35.96% and 63.54%, respectively.

[0038] Implementation Example 7

[0039] Mix 30g defatted wheat germ powder and 300mL water, shake on a shaker at 30℃ for 1.5h, centrifuge the extract at 8000r / min for 20min at 4℃, take the supernatant and freeze-dry. Adjust the substrate concentration to 2%, the amount of papain added to 8000U / g, and the pH to 5.0. Place the resulting enzymatic hydrolysis system solution at 45℃, 50℃, 55℃, 60℃, and 65℃ for 2h. Immediately after the reaction, raise the temperature of the enzymatic hydrolysis system to 100℃ to inactivate the enzyme for 10min to terminate the reaction, and obtain an enzymatic hydrolysis peptide solution with antioxidant properties. The powdered anti-fatigue peptide was freeze-dried. The ABTS free radical scavenging activity of the five peptide segments and wheat germ albumin was determined, and VC was used as a positive control. Figure 7 As shown, the ABTS free radical scavenging rates were 32.937%, 92.52%, 99.217%, 97.143%, 98.67%, 76.6% and 99.48%, respectively.

[0040] Implementation Example 8

[0041] Mix 30g defatted wheat germ powder and 300mL water, shake on a shaker at 30℃ for 1.5h, centrifuge the extract at 8000r / min for 20min at 4℃, take the supernatant and freeze-dry. Adjust the substrate concentration to 2%, the amount of papain added to 8000U / g, and the pH to 5.0. Place the resulting enzymatic hydrolysis system solution at 45℃, 50℃, 55℃, 60℃, and 65℃ for 2h. Immediately raise the temperature of the enzymatic hydrolysis system to 100℃ to inactivate the enzyme for 10min to terminate the reaction, and obtain an enzymatic hydrolysis peptide solution with antioxidant properties. The powdered anti-fatigue peptide was freeze-dried to determine the hydroxyl radical scavenging activity of the five peptide segments and wheat germ albumin, and VC was used as a positive control. Figure 8 As shown, the hydroxyl radical scavenging rates were 33.113%, 29.453%, 81.533%, 70.763%, 57.25%, 28.843% and 97.813%, respectively.

[0042] Implementation Example 9

[0043] Mix 30g defatted wheat germ powder and 300mL water, shake on a shaker at 30℃ for 1.5h, centrifuge the extract at 8000r / min for 20min at 4℃, take the supernatant and freeze-dry. Adjust the substrate concentration to 2%, the amount of papain added to 8000U / g, and the pH to 5.0. Place the resulting enzymatic hydrolysis system solution at 45℃, 50℃, 55℃, 60℃, and 65℃ for 2h. Immediately after the reaction, raise the temperature of the enzymatic hydrolysis system to 100℃ to inactivate the enzyme for 10min to terminate the reaction, and obtain an enzymatic hydrolysis peptide solution with antioxidant properties. The powdered anti-fatigue peptide was freeze-dried. The Fe2+ chelating ability of the five peptide segments and wheat germ albumin was determined, and VC was used as a positive control. Fig. 9 As shown, the Fe2+ chelating capacities are 3.067%, 3.267%, 4.033%, 2.5%, -0.387%, -3.243%, and 12.123%, respectively.

[0044] Anti-fatigue peptides mainly target the scavenging properties of hydroxyl radicals, DPPH radicals, Fe2+ chelating ability and ABTS radicals.

[0045] The anti-fatigue peptide prepared according to the method of the present invention can be applied to the base material of natural food preservatives and the ingredients of functional antioxidant foods. The obtained anti-fatigue peptide has a strong free radical scavenging effect.

[0046] Taking the free radical scavenging activity of the enzymatic system as an indicator, papain is used to hydrolyze wheat germ albumin, and separation techniques such as ultrafiltration are used to prepare peptides with strong antioxidant properties.

[0047] Take a certain amount of defatted wheat germ powder, mix it with deionized water at a solid-liquid ratio of 1:10 (W / V), shake it on a shaker at 30℃ for 1.5h, centrifuge the extract at 8000r / min for 20min at 4℃, take the supernatant and freeze-dry it; optimize the enzymatic hydrolysis conditions, find the best enzymatic hydrolysis conditions, stir and adjust the pH value of the mixed solution to 6, add papain, the enzyme amount is 8000U / g, the substrate concentration is 2%, put it in a 55℃ water bath for 2h, and immediately raise the temperature of the enzymatic hydrolysis system to 100℃ to kill the enzyme for 10min to terminate the reaction, obtain an enzymatic hydrolysis peptide liquid with antioxidant properties, and ultrafilter the enzymatic hydrolysis peptide liquid to obtain peptides in five molecular weight ranges, freeze-dry the powdered anti-fatigue peptides, and measure the antioxidant activity of each peptide segment. The results show that the peptide segments in the molecular weight range of 3-5kDa have obvious effects in scavenging hydroxyl radicals, DPPH radicals, and ABTS radicals. It is highly safe for consumption.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing anti-fatigue peptides from wheat germ albumin by enzymatic hydrolysis, characterized in that: This method controls the enzymatic hydrolysis reaction conditions and ultrafilters the enzymatic peptide solution to obtain peptide segments with better free radical scavenging effects. The specific steps are as follows: (1) Take a certain amount of defatted wheat germ powder, mix it with deionized water at a solid-liquid ratio of 1:10 (W / V), shake it on a shaker at 30°C for 1.5 h, centrifuge the extract at 8000 r / min for 20 min at 4°C, take the supernatant and freeze-dry it; (2) adjusting the pH value of the mixed solution to 6 by stirring, adding papain at an enzyme amount of 8000 U / g and a substrate concentration of 2%; (3) placing the mixture in a water bath at 55°C for 2 h, and immediately raising the temperature of the enzymatic hydrolysis system to 100°C for 10 min to inactivate the enzyme and obtain an enriched reaction solution containing anti-fatigue peptides. (4) The enzymatic peptide solution was passed through ultrafiltration membranes of 10KDa, 5KDa, 3KDa and 1KDa to obtain five peptide fragments: MW>10kDa, MW5–10kDa, MW3–5kDa, MW1–3kDa and MW<1kDa; (5) The free radical scavenging activity of the five peptides was measured to find the peptide with better antioxidant properties.

2. The method for preparing anti-fatigue peptides from wheat germ albumin by enzymatic hydrolysis according to claim 1, characterized in that: The protein source of the peptide is the wheat germ byproduct after defatting wheat germ.

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