A method for preparing functional peptides using ginger extract and wheat gluten powder

Abstract

The application provides a method for preparing functional peptides by using ginger extract and wheat gluten powder and application, and the ginger extract is prepared by using a phosphate buffer solution containing Cys, the obtained ginger extract has high ginger protease enzyme activity, and the enzyme hydrolysis reaction is carried out under the control of the ginger extract and the wheat gluten powder, so that the high-efficiency preparation of wheat functional peptides with DPP-IV inhibitory activity and antioxidant property can be realized.

Description

Technical Field

[0001] This invention belongs to the field of plant protein deep processing technology, specifically involving a method for preparing functional peptides using ginger extract and gluten powder. Background Technology

[0002] Many common diseases, such as diabetes mellitus (DM), cardiovascular disease, and chronic kidney disease, are associated with common complications such as inflammation, and there is a close interaction between inflammation and oxidative stress. The large amount of reactive oxygen species produced by cells during physiological activities leads to an imbalance in the body's oxidation-antioxidant balance, further causing oxidative stress and ultimately triggering inflammation.

[0003] Diabetes mellitus (DM) is a metabolic disease associated with hyperglycemia, classified into insulin-dependent diabetes mellitus (Type 1) and non-insulin-dependent diabetes mellitus (Type 2). Type 2 diabetes mellitus (T2DM) is a complex disease caused by insufficient insulin production or the body's inability to utilize its own insulin. The number of people with T2DM is increasing annually, accounting for approximately 93% of all diabetes cases. Currently, treatment for T2DM primarily focuses on inhibiting starch absorption in the intestinal lumen, controlling blood glucose levels, increasing the body's sensitivity to insulin, and reducing the damage caused by hyperglycemia. Dipeptidyl peptidase IV (DPP-IV) is a serine protease widely distributed in human tissues. It can degrade and inactivate the intestinal glucagon hormone glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP). The latest treatment for T2DM involves using DPP-IV inhibitors to prevent the loss of GLP-1 and GIP activity. Clinically, chemically synthesized DPP-IV inhibitors such as sitagliptin are used to treat type 2 diabetes, but these drugs are expensive and have side effects. Therefore, research on extracting active ingredients with DPP-IV inhibitory effects from natural proteins is of great significance.

[0004] Wheat gluten, as a product of deep wheat processing, contains approximately 70% protein (wheat gluten protein). Its content of wheat gluten protein Pro is as high as 13%, and its unique amino acid composition and potential bioactive peptide sequence give it excellent potential for preparing DPP-IV inhibitory peptides. Furthermore, reports indicate that wheat gluten protein hydrolysates also possess antioxidant, blood pressure-lowering, and immunomodulatory effects.

[0005] Ginger is a natural spice and food supplement, rich in gingerol and gingerone. Studies have shown that it possesses a wide range of functional activities, such as antioxidant and anti-inflammatory properties. Furthermore, reports indicate that ginger extract can inhibit α-amylase and α-glucosidase, thereby preventing carbohydrate digestion and reducing glucose production.

[0006] This invention combines the advantages of ginger and gluten powder. By preparing a ginger extract containing ginger protease and mixing it with gluten powder, the ginger protease further hydrolyzes wheat gluten protein, thereby improving the solubility of gluten powder and preparing functional wheat peptides with DPP-IV inhibitory activity and antioxidant properties.

[0007] This invention prepares functional peptides with DPP-IV inhibitory and antioxidant activities through enzymatic hydrolysis, which not only helps to broaden the application range of wheat gluten protein and ginger and improve their nutritional value, but also helps to promote the development of hypoglycemic and antioxidant foods. Summary of the Invention

[0008] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0009] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0010] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing functional peptides using ginger extract and gluten powder.

[0011] To solve the above-mentioned technical problems, the present invention provides the following technical solution: including,

[0012] Preparation of ginger extract: Ginger is mixed with phosphate buffer solution and homogenized, then extracted, filtered and the filtrate is collected; phosphate buffer solution is added to the residue and the extraction is repeated, then the filtrate is collected after filtration; the above filtrates are combined to obtain ginger extract;

[0013] Protein hydrolysis: Gluten powder is dispersed in the above ginger extract for enzymatic hydrolysis. After hydrolysis, the enzyme is inactivated, the supernatant is collected by centrifugation, and the supernatant is desalted and dried to obtain functional peptide powder.

[0014] As a preferred embodiment of the method for preparing functional peptides using ginger extract and gluten powder according to the present invention, wherein: the phosphate buffer solution is a phosphate buffer solution containing Cys, wherein the phosphate concentration is 0.01-0.1M and the Cys concentration is 5-10mM.

[0015] In a preferred embodiment of the method for preparing functional peptides using ginger extract and gluten powder as described in this invention, the pH of the phosphate buffer solution is 5.5–7.0.

[0016] In a preferred embodiment of the method for preparing functional peptides using ginger extract and gluten powder as described in this invention, the ratio of ginger to phosphate buffer solution is 1:3 to 1:5.

[0017] As a preferred embodiment of the method for preparing functional peptides using ginger extract and gluten powder according to the present invention, the mixing and homogenization is performed at 10,000-20,000 r / min for 3-8 min, and the extraction time is 20-40 min.

[0018] As a preferred embodiment of the method for preparing functional peptides using ginger extract and gluten powder according to the present invention, wherein the total activity of ginger protease in the ginger extract is 32000-42000U.

[0019] As a preferred embodiment of the method for preparing functional peptides using ginger extract and gluten powder according to the present invention, the gluten powder is dispersed in the above-mentioned ginger extract for enzymatic hydrolysis, wherein the mass concentration of gluten powder is 1-5% and the concentration of ginger protease is 1500-2500 U / g gluten powder.

[0020] As a preferred embodiment of the method for preparing functional peptides using ginger extract and gluten powder according to the present invention, the enzymatic hydrolysis is performed at a pH of 5.5–7.0, for a time of 3–6 h, and at a temperature of 55–65 °C.

[0021] As a preferred embodiment of the method for preparing functional peptides using ginger extract and gluten powder according to the present invention, the protein recovery rate of the functional peptides prepared by the method is greater than 70%; the proportion of peptides with a molecular weight of less than 1000 Da is greater than 70%; the inhibition rate of dipeptidyl peptidase IV is greater than 50%; and the antioxidant activity is greater than 40%.

[0022] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application for preparing functional peptides using ginger extract and gluten powder.

[0023] As a preferred embodiment of the application of the functional peptides prepared by ginger extract and gluten powder according to the present invention, the application includes using the functional peptides in the preparation of hypoglycemic products or antioxidant products.

[0024] Beneficial effects of this invention:

[0025] (1) The ginger extract prepared by the present invention has good ginger protease activity. It has specific enzyme cleavage sites and can efficiently prepare DPP-Ⅳ inhibitory active peptides. At the same time, the ginger extract is directly used to enzymatically hydrolyze wheat gluten protein, avoiding the cumbersome protease separation and purification process and the loss of total enzyme activity during purification, and effectively utilizing the activity of ginger protease.

[0026] (2) The preferred plant raw material of the present invention, wheat gluten protein, has a unique amino acid composition and its potential active peptide sequence, which provides a guarantee for the effective acquisition of functional peptides.

[0027] (3) This invention makes full use of the advantages of ginger and gluten powder, combines the specificity of protease with the specificity of substrate amino acid composition and sequence, and achieves the preparation of functional peptides by optimizing the enzymatic hydrolysis conditions. Detailed Implementation

[0028] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0029] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0030] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0031] The analysis methods used in the embodiments of this invention are as follows:

[0032] 1. Determination of protein content: Semi-micro Kjeldahl method (GB5009.5-2016);

[0033] 2. Determination of protein recovery rate of wheat peptide powder: Protein recovery rate of wheat peptide powder (%) = Protein content of wheat peptide powder × 100 / Protein content of raw material before enzymatic hydrolysis;

[0034] 3. Determination of peptide molecular weight: The molecular weight distribution of the prepared wheat peptide product was determined by gel permeation chromatography (GPC). The sample was prepared with a wheat peptide concentration of 1.8 mg / mL, centrifuged at 10000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm cellulose acetate microporous membrane. 20 μL of the filtrate was injected. A TSK-gel G2000 SWXL column (7.8 mm × 300 mm) was used for analysis, and the column was equilibrated with a mobile phase (acetonitrile, water, TFA volume ratio of 45:55:0.1). The detection wavelength and column temperature were 214 nm and 25 °C, respectively, and the mobile phase flow rate was 0.5 mL / min. Molecular weight distribution standards were used: cytochrome C (12500 Da), antimicrobial peptide (6500 Da), bacitracin (1450 Da), GGA T (450 Da), and GGG (189 Da).

[0035] 4. Determination of dipeptidyl peptidase IV inhibitory activity: In a 96-well microplate, 25 μL of sample (dissolved in pH 8.0, 100 mM Tris-HCl buffer, protein concentration of enzyme digest 1.8 mg / mL) was mixed with 25 μL of Gly-Pro-PNA (1.6 mM), incubated at 37°C for 10 min, then 50 μL of DPP-IV (10 U / L) solution was added to initiate the reaction, and the reaction was carried out at 37°C for 60 min. Then, 100 μL of sodium acetate buffer (1 M, pH 4.0) was added to terminate the reaction, and the absorbance was measured at 405 nm. A blank control group was prepared using Tris-HCl buffer instead of DPP-IV solution, and a control group was prepared using Tris-HCl buffer instead of the sample. The DPP-IV inhibition rate was calculated using the following formula:

[0036]

[0037] In the formula, A sample: OD during the normal reaction of DPP-IV, substrate Gly-Pro-pNA, and repressor peptide sample. 405 ;

[0038] Sample blank A: Replace the OD of the DPP-IV reaction with Tris-HCl buffer. 405 ;

[0039] A negative: Use Tris-HCl buffer instead of the OD after the peptide reaction. 405 ;

[0040] A negative blank: Replace DPP-IV and the inhibitory peptide with Tris-HCl buffer. 405 .

[0041] 5. Determination of antioxidant activity:

[0042] DPPH free radical scavenging activity assay: In a 96-well microplate, 100 μL of wheat enzyme hydrolysate (1.8 mg / mL) was mixed with an equal volume of 0.2 mM DPPH solution and reacted at room temperature in the dark for 30 min. The absorbance was then read at 517 nm using a microplate reader (Bio-Rad).

[0043]

[0044] In the formula: A: is the OD of the sample after the reaction with DPPH. 517 ;

[0045] Blank A: OD of ethanol instead of DPPH 517 ;

[0046] Control A: OD of the sample was replaced with ethanol. 517 .

[0047] Example 1

[0048] Preparation of ginger extract:

[0049] Add 60g of peeled and chopped ginger to 240mL of phosphate buffer solution (0.01M, pH 6.5, containing 10mM Cys), homogenize at 20000r / min for 5min, stir and extract for 40min, filter through four layers of gauze, and collect the filtrate.

[0050] Add 120 mL of the above phosphate buffer solution to the residue, stir and extract again for 40 min, filter through four layers of gauze, and collect the filtrate; combine the filtrates to obtain ginger extract;

[0051] The total activity of ginger protease in the extract was determined to be 4.07 × 10⁻⁶. 4 U.

[0052] Protein hydrolysis:

[0053] The gluten powder was dispersed in ginger extract at a ratio of 1500 U ginger protease / g gluten powder to achieve a gluten powder mass concentration of 5%. The mixture was enzymatically hydrolyzed at pH 6.5 and 60℃ for 360 min. After the enzymatic hydrolysis, the enzyme was inactivated in a 95℃ water bath for 5 min. The mixture was then cooled, centrifuged at 8000g for 30 min, and the supernatant was collected. The supernatant was then desalted, concentrated, and dried to prepare functional peptide powder.

[0054] The functional peptide powder prepared by the method in this embodiment has a protein recovery rate of 72.10%, a peptide fraction with a molecular weight of less than 1000 Da of 86.67%, a DPP-Ⅳ inhibition rate of 56.35%, and an antioxidant activity of 42.33%.

[0055] Example 2

[0056] Preparation of ginger extract:

[0057] Add 60g of peeled and chopped ginger to 240mL of phosphate buffer solution (0.01M, pH 5.5, containing 10mM Cys), homogenize at 20000r / min for 5min; stir and extract for 20min, filter through four layers of gauze, and collect the filtrate.

[0058] Add 120 mL of the above phosphate buffer solution to the residue, stir and extract again for 20 min, filter through four layers of gauze, and collect the filtrate; combine the filtrates to obtain ginger extract;

[0059] The total activity of ginger protease in the extract was determined to be 3.63 × 10⁻⁶. 4 U.

[0060] Protein hydrolysis:

[0061] The gluten powder was dispersed in ginger extract at a ratio of 2000 U ginger protease / g gluten powder to achieve a gluten powder mass concentration of 3%. The mixture was hydrolyzed at pH 5.5 and 55℃ for 300 min. After the enzymatic hydrolysis was completed, the enzyme was inactivated in a 95℃ water bath for 5 min. After cooling, the mixture was centrifuged at 8000g for 30 min. The supernatant was collected, desalted, concentrated, and dried to obtain functional peptide powder.

[0062] The functional peptide powder prepared by the method in this embodiment has a protein recovery rate of 76.01%, peptides with a molecular weight of less than 1000 Da account for 82.27%, a DPP-Ⅳ inhibition rate of 51.29%, and an antioxidant activity of 50.70%.

[0063] Example 3

[0064] Preparation of ginger extract:

[0065] Add 60g of peeled and chopped ginger to 240mL of phosphate buffer solution (0.1M, pH 7.0, containing 10mM Cys), homogenize at 16000r / min for 5min, stir and extract for 20min, filter through four layers of gauze, and collect the filtrate.

[0066] Add 120 mL of the above phosphate buffer solution to the residue, stir and extract again for 20 min, filter through four layers of gauze, and collect the filtrate; combine the filtrates to obtain ginger extract;

[0067] The total activity of ginger protease in the extract was determined to be 3.50 × 10⁻⁶. 4 U.

[0068] Protein hydrolysis:

[0069] The gluten powder was dispersed in ginger extract at a ratio of 2500 U ginger protease / g gluten powder to achieve a gluten powder mass concentration of 2%. The mixture was hydrolyzed at pH 7.0 and 65℃ for 240 min. After enzymatic hydrolysis, the enzyme was inactivated in a 95℃ water bath for 5 min. The mixture was then cooled, centrifuged at 8000g for 30 min, and the supernatant was collected. The supernatant was then desalted, concentrated, and dried to obtain functional peptide powder.

[0070] The functional peptides prepared by the method in this embodiment have a protein recovery rate of 80.28%, a molecular weight of less than 1000 Da accounting for 73.07%, a DPP-IV inhibition rate of 50.51%, and an antioxidant activity of 56.59%.

[0071] Comparative Example 1

[0072] Preparation of ginger aqueous extract:

[0073] Mix 60g of peeled and chopped ginger with 240mL of water, homogenize at 20000r / min for 5min, stir and extract for 20min, and filter through four layers of gauze.

[0074] Add 120 mL of the above water to the residue, stir and extract again for 20 min, filter through four layers of gauze, and collect the filtrate; combine the filtrates to obtain ginger extract.

[0075] The total activity of ginger protease in the extract was determined to be 0.14 × 10⁻⁶. 4 U.

[0076] It is evident that the ginger aqueous extract prepared by this comparative method has low activity, which is insufficient to meet the requirements for further enzymatic hydrolysis of gluten.

[0077] Comparative Example 2

[0078] Gluten powder was directly dispersed in water to make the gluten powder concentration of the suspension 5%. The suspension was heated at pH 6.5 and 60℃ for 360 min. After enzymatic hydrolysis, the suspension was heated in a 95℃ water bath for 5 min. After cooling, the suspension was centrifuged at 8000g for 30 min to obtain the supernatant. The supernatant was then desalted, concentrated and dried to obtain peptide powder.

[0079] The peptide powder prepared by this comparative method had a protein recovery rate of 5.03%, and peptides with a molecular weight of less than 1000 Da accounted for approximately 49.29% of the total peptides; the DPP-IV inhibition rate was 7.74%, and the antioxidant activity was 16.38%.

[0080] Table 1. Relevant Measurement Indicators in Functional Peptide Preparation Process

[0081]

[0082] As can be seen from the above embodiments and comparative examples:

[0083] Compared to the preparation in Comparative Example 1, the ginger aqueous extract obtained had lower activity, with a total enzyme activity of only 0.14 × 10⁻⁶. 4 U is insufficient to meet the requirements for further enzymatic hydrolysis of gluten powder. The ginger protein extract prepared using the methods in Examples 1-3 of this invention has a total enzyme activity as high as 3.5-4.07×10⁻⁶. 4 U significantly improves the extraction rate and activity retention rate of ginger protease, resulting in higher efficiency.

[0084] Compared to the protein recovery rate of only 5.03% in the method for preparing functional peptides in Comparative Example 2, the protein recovery rate of Examples 1-3 of this invention was significantly improved—between 72.10% and 80.28%, and the content of peptides with a molecular weight of less than 1000 Da was also greatly improved—from 49.29% to 86.67%.

[0085] Meanwhile, the DPP-Ⅳ inhibitory activity of Comparative Example 2 was only 7.74%, while the DPP-Ⅳ inhibitory activity of Examples 1-3 of this invention was significantly improved, with inhibitory activities ranging from 50.51% to 56.35%, IC50... 50 It can reach 1.29 mg / mL, and its antioxidant activity can reach 56.59%.

[0086] Therefore, the method for preparing functional peptides by enzymatic hydrolysis of wheat gluten protein using an extract containing ginger protease provided by the present invention has significant advantages, including high protein recovery rate, high content of low molecular weight wheat peptides in the product, and good hypoglycemic and antioxidant activity.

[0087] Example 4

[0088] To further explore the beneficial effects of the phosphate buffer extraction of ginger protease in this invention, this embodiment investigated the effects of buffer pH and Cys concentration on the technical effect, and the results are shown in Tables 2 and 3.

[0089] A. The pH of the phosphate buffer was adjusted as shown in Table 2. The rest of the preparation process was the same as in Example 1. The results are shown in Table 2.

[0090] Table 2. Effect of phosphate buffer pH on total protease activity in ginger extract.

[0091]

[0092] As shown in Table 2, the total enzyme activity was ≥3.5×10⁻⁶ when the pH was between 5.5 and 7.0. 4 U, among which the total enzyme activity of ginger protease was highest at pH 6.5, reaching 4.07 × 10⁻⁶. 4 U.

[0093] B. Adjust the concentration of Cys in the phosphate buffer as shown in Table 3. The rest of the preparation process is the same as in Example 1. The results are shown in Table 3.

[0094] Table 3. Effect of Cys concentration on the total protease activity of ginger extract.

[0095]

[0096] As shown in Table 3, compared to the total enzyme activity of 10 mM Cys ginger extract (4.07 × 10⁻⁶), the total enzyme activity of 10 mM Cys ginger extract was significantly lower. 4 The total enzyme activity of ginger extract with low Cys concentrations (0–1 mM) was low, with a protein recovery rate of 72.1%. In particular, the total enzyme activity of ginger protease was only 0.10 × 10⁻⁶ when the Cys concentration was 0 mM. 4 U, which cannot meet the activity requirements for further enzymatic hydrolysis to prepare wheat peptides; while the total enzyme activity and protein recovery rate of ginger extract at high Cys content (15mM) showed only a small increase (4.11×10). 4 U and 74.3%), therefore a feed-to-liquid ratio of 10mM is more suitable for production.

[0097] Example 5

[0098] To investigate the benefits of using ginger protease to hydrolyze wheat gluten protein in this invention, different types of proteases were used in this embodiment. Except for the different optimal pH and temperature of the different enzymes, the specific hydrolysis process was the same as in Example 1. The results are shown in Table 4.

[0099] Table 4. Effects of protease type on wheat gluten protein hydrolysate

[0100]

[0101] As shown in Table 4, compared with the enzymatic hydrolysis of wheat gluten with ginger extract, the DPP-IV inhibitory activity and antioxidant activity of other commercial protease hydrolysates were significantly lower than those of ginger extract hydrolysate. This indicates that the present invention has significant advantages in preparing functional peptides by enzymatic hydrolysis of wheat gluten with ginger extract.

[0102] This invention prepares ginger extract using a phosphate buffer containing Cys, resulting in extract with high ginger protease activity. By utilizing the ginger extract and gluten powder under controlled enzymatic hydrolysis conditions, efficient preparation of wheat functional peptides with DPP-IV inhibitory activity and antioxidant properties can be achieved.

[0103] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing functional peptides using ginger extract and gluten powder, characterized in that: include, Preparation of ginger extract: Ginger was mixed with phosphate buffer solution and homogenized, then extracted, filtered and the filtrate was collected; phosphate buffer solution was added to the residue and the extraction was repeated, then the filtrate was collected; the above filtrates were combined to obtain ginger extract with a total ginger protease activity of 32000~42000 U; The mixing and homogenizing process is carried out at 10,000-20,000 r / min for 5 min, and the extraction time is 20-40 min. The ratio of ginger to phosphate buffer solution is 1:3 to 1:5 g / mL; The phosphate buffer solution is a phosphate buffer solution containing Cys, wherein the phosphate concentration is 0.01~0.1 M, the Cys concentration is 10 mM, and the pH is 5.5~7.0; Protein hydrolysis: Gluten powder is dispersed in the above ginger extract for enzymatic hydrolysis. After hydrolysis, the enzyme is inactivated, the supernatant is collected by centrifugation, and the supernatant is desalted and dried to obtain functional peptide powder. The concentration of gluten powder is 1-5%, the concentration of ginger protease is 1500-2500 U / g gluten powder, the enzymatic hydrolysis pH is 5.5-7.0, the enzymatic hydrolysis time is 3-6 h, and the enzymatic hydrolysis temperature is 55-65 ℃. The protein recovery rate of the functional peptides prepared by the method is greater than 70%; of which, the proportion of peptides with a molecular weight of less than 1000 Da is greater than 70%; the inhibition rate of dipeptidyl peptidase IV is greater than 50%; and the antioxidant activity is greater than 40%.

2. An application of a functional peptide prepared by the method described in claim 1, characterized in that: The applications include using the functional peptides in the preparation of hypoglycemic or antioxidant products.

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