Antioxidant peptide derived from gastrointestinal digests of waxy corn and application of antioxidant peptide
By preparing the antioxidant peptides extracted from the gastrointestinal digesta of glutinous corn, the research gap of the antioxidant active peptides of glutinous corn and the problem of side effects of synthetic antioxidants are solved, providing an efficient natural antioxidant resource for application in food and medicine.
Patent Information
- Application Number
- CN202511022818.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-23
- Publication Date
- 2025-10-17
AI Technical Summary
Existing research has failed to effectively utilize the antioxidant active peptides released by glutinous corn during gastrointestinal digestion, and synthetic antioxidants have side effects in food, and there is a lack of natural, non-toxic alternatives.
By simulating the human digestion process, the gastrointestinal digest of glutinous corn was prepared, and antioxidant peptides with amino acid sequences of DEC, IGW, GSW, CCHQI, RQPQCSP, LRQPQCSP and FAGC were extracted for use in food and medicine.
It provides an efficient natural antioxidant resource, solves the problems of high-value utilization of glutinous corn and food safety, and has significant antioxidant activity.
Smart Images
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of antioxidant peptides, and particularly relates to an antioxidant peptide derived from waxy corn gastrointestinal digesta and application thereof. BACKGROUND
[0002] The flavor, color and ingredients of food can be changed due to oxidation, and the food can be seriously deteriorated. Some synthetic antioxidants commonly used in food at present include butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tertiary butylhydroquinone (TBHQ) and the like, although the effect is significant, but there are certain side effects in the application of food. The development of natural and non-toxic antioxidant has important significance for food storage development and human health. The development of antioxidant has become one of the current research hotspots.
[0003] Antioxidant peptides are polypeptides or small peptides with antioxidant activity generated by enzymatic hydrolysis of proteins, which have the characteristics of wide source, high safety, etc., and can replace synthetic antioxidants and be applied in the field of food.
[0004] Plant proteins have always been the main source of antioxidant peptides, such as corn protein, soybean protein, pea protein, rice protein, etc. Waxy corn, as an important branch of corn, is an important delicacy on people's table. Although there are many articles reporting that antioxidant active peptides can be obtained from corn protein by enzymatic hydrolysis, the existing research on waxy corn mainly focuses on the analysis of nutritional components and the optimization of processing technology, and the research on whether antioxidant active peptides are released from waxy corn during gastrointestinal digestion is still in a blank state. SUMMARY
[0005] Therefore, one of the purposes of the present application is to provide an antioxidant peptide derived from waxy corn gastrointestinal digesta, which has strong antioxidant activity and can be used as an antioxidant in the food industry.
[0006] The second purpose of the present application is to provide a waxy corn gastrointestinal digesta containing the antioxidant peptide and a preparation method thereof.
[0007] In order to achieve the above-mentioned purposes, the present application provides the following technical solutions.
[0008] The present application provides an antioxidant peptide derived from waxy corn gastrointestinal digesta, wherein the antioxidant peptide comprises at least one of the amino acid sequences DEC, IGW, GSW, CCHQI (SEQ ID NO. 1), RQPQCSP (SEQ ID NO. 2), LRQPQCSP (SEQ ID NO. 3) and FAGC (SEQ ID NO. 4).
[0009] The application further provides a waxy corn gastrointestinal digestate, comprising the antioxidant peptide.
[0010] The application further provides a preparation method of the waxy corn gastrointestinal digestate, comprising the following steps: after the waxy corn is cooked, the waxy corn is crushed to obtain a simulated oral chew; the simulated oral chew is mixed with simulated saliva, an α-amylase solution and a CaCl2 solution, and stirred at 37°C to obtain an oral digestate; the pH of the oral digestate is adjusted to 3.0, and then the oral digestate is mixed with a pepsin solution, simulated gastric juice and a CaCl2 solution, and stirred at 37°C for 2 hours to obtain a gastric digestate; the gastric digestate is mixed with simulated intestinal juice and a CaCl2 solution, the pH is adjusted to 7.0, and then a trypsin solution is added, and the mixture is stirred at 37°C for 2 hours, and then centrifuged to inactivate the enzyme, and the supernatant is obtained to obtain the gastrointestinal digestate.
[0011] The application is not particularly limited to the specific sources of the raw materials. In the application, the simulated saliva is preferably composed of the following raw materials according to volume fractions: 0.5 mol / L of KCl 15.1 parts, 0.5 mol / L of KH2PO4 3.7 parts, 1 mol / L of NaHCO3 6.8 parts, 0.15 mol / L of MgCl2 0.5 parts and 0.5 mol / L of (NH4)2CO3 0.06 parts. The simulated gastric juice is preferably composed of the following raw materials according to volume fractions: 0.5 mol / L of KCl 6.9 parts, 0.5 mol / L of KH2PO4 0.9 parts, 1 mol / L of NaHCO3 12.5 parts, 2 mol / L of NaCl 11.8 parts, 0.15 mol / L of MgCl2 0.4 parts and 0.5 mol / L of (NH4)2CO3 0.5 parts. The simulated intestinal juice is preferably composed of the following raw materials according to volume fractions: 0.5 mol / L of KCl 6.8 parts, 0.5 mol / L of KH2PO4 0.8 parts, 1 mol / L of NaHCO3 32.5 parts, 2 mol / L of NaCl 9.6 parts and 0.15 mol / L of MgCl2 1.1 parts.
[0012] In the present application, the concentration of the CaCl2 solution is preferably 0.3 mol / L; the weight volume ratio of the simulated oral chew to the simulated saliva, the alpha-amylase solution and the CaCl2 solution is preferably 10 g:8.95 mL:1 mL:50 μL; the concentration of the alpha-amylase solution is preferably 37 U / mL. The weight volume ratio of the oral chew to the pepsin solution, the simulated gastric juice and the CaCl2 solution is preferably 10 g:2 mL:17.99 mL:10 μL, and the concentration of the pepsin solution is preferably 1800 U / mL. The weight volume ratio of the oral chew to the simulated intestinal fluid, the CaCl2 solution and the trypsin solution is preferably 5 g:17.99 mL:10 μL:2 mL, and the concentration of the trypsin solution is preferably 250 U / mL.
[0013] The present application also provides the use of the above-mentioned antioxidant peptide, the above-mentioned waxy maize gastrointestinal digest or the above-mentioned preparation method in the preparation of an antioxidant product. In the present application, the type of the product preferably includes food and / or medicine.
[0014] The present application has the following beneficial effects:
[0015] The present application first identifies an antioxidant peptide with high antioxidant activity from the in vitro gastrointestinal digest of waxy maize, which not only provides a theoretical basis for the high-value utilization of waxy maize, but also provides a new resource and technical path for the development of natural antioxidants, and has important significance for the research and development of antioxidant products. DETAILED DESCRIPTION
[0016] The technical solutions provided by the present application will be described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.
[0017] In the following examples, all are conventional methods unless otherwise specified.
[0018] In the following examples, the materials, reagents, etc. used are commercially available unless otherwise specified.
[0019] In the following examples, the waxy maize is purchased from Beidahuang Food Co., Ltd., the alpha-amylase is purchased from Shanghai Senye Biotechnology Co., Ltd., the pepsin is purchased from Shanghai Senye Biotechnology Co., Ltd., and the trypsin is purchased from Shanghai Senye Biotechnology Co., Ltd.
[0020] Example 1
[0021] A waxy maize gastrointestinal digest is prepared by the following method:
[0022] Waxy corn was heated in boiling water at standard atmospheric pressure for 25 min, and after cooling, the kernels were stripped and crushed using a cell disrupter to obtain simulated oral chew. 10 g of simulated oral chew was mixed with 8.95 mL of simulated saliva, 1 mL of α-amylase solution (the concentration of the α-amylase solution was 37 U / mL), 50 μL of CaCl2solution (the concentration of the CaCl2solution was 0.3 mol / L), and placed in a magnetic stirrer, heated and stirred at 37°C for 5 min to obtain oral digesta.
[0023] The pH of the oral digesta was adjusted to 3.0 using 1 mol / mL HCl, and then 2 mL of pepsin solution (the concentration of the pepsin solution was 1800 U / mL) was added, 17.99 mL of simulated gastric juice and 10 μL of CaCl2solution (the concentration of the CaCl2solution was 0.3 mol / L) were added, and after mixing, it was placed in a magnetic stirrer and heated and stirred at 37°C for 2 h to obtain gastric digesta.
[0024] 35.98 mL of simulated intestinal fluid and 20 μL of CaCl2solution (the concentration of the CaCl2solution was 0.3 mol / L) were added to the gastric digesta, and the pH was adjusted to 7.0 with NaOH (1 mol / L), and then 4 mL of trypsin solution (the concentration of the trypsin solution was 250 U / mL) was added, and the mixture was heated and stirred at 37°C for 2 h to simulate intestinal digestion. After the reaction was completed, the enzyme was inactivated by boiling water for 10 min, and centrifuged at 4°C and 4000 r / min for 10 min. The supernatant was collected to obtain the gastro-intestinal digesta, which was freeze-dried for later use.
[0025] The compositions of the simulated saliva, simulated gastric juice and simulated intestinal fluid used in the above process are shown in Table 1.
[0026] Table 1 Composition of simulated saliva, simulated gastric juice and simulated intestinal fluid
[0027] Mock saliva (mL) Mock gastric fluid (mL) Mock intestinal fluid (mL) KCl (0.5 mol / L) 15.1 6.9 6.8 KH2PO4 (0.5 mol / L) 3.7 0.9 0.8 NaHCO3(1 mol / L) 6.8 12.5 42.5 NaCl (2 mol / L) - 11.8 9.6 MgCl2 (0.15 mol / L) 0.5 0.4 1.1 (NH4)2CO3 (0.5 mol / L) 0.06 0.5 -
[0028] Example 2
[0029] Mass spectrometric identification of the gastro-intestinal digesta obtained in Example 1
[0030] The waxy corn gastrointestinal digest was subjected to LC-MS / MS mass spectrometry identification to determine its polypeptide composition. 1 mL of the gastrointestinal digest sample was lyophilized and re-dissolved with 100 μL of NH4HCO3(50 mmol / L) solution. 1 μL of DTT (1 mmol / L) solution was accurately pipetted into the sample to make the final concentration of DTT 10 mmol / L, and the sample was subjected to reduction reaction at 56°C water bath for 1 h. 2 μL of iodoacetamide (IAM) solution (1 mmol / L) was accurately pipetted into the sample to make the final concentration of IAM 20 mmol / L, and the sample was continuously reacted at room temperature in the dark for 40 min. Then, 1 μL of DTT (1 mmol / L) solution was accurately pipetted into the sample to make the final concentration of DTT 10 mmol / L to neutralize the unreacted IAA. The above reaction solution was subjected to desalting treatment with a C18 stage-tip chromatographic column, and then was dried under vacuum at 45°C. The dried sample was dissolved and subjected to mass spectrometry analysis, and the amino acid sequence of the identified peptide was determined by comparison with the corn protein data.
[0031] Prediction and screening of the activity of the peptide: the potential biological activity of the identified peptide was calculated by the Peptide Ranker online software, and the polypeptide components with a prediction value greater than 0.5 were screened. Combined with the content of each polypeptide in the mass spectrometry results, the top 7 peptides in the comprehensive ranking were selected for solid-phase synthesis of the peptides. The solid-phase synthesized peptide sequences were DEC, IGW, GSW, CCHQI, RQPQCSP, LRQPQCSP and FAGC, respectively.
[0032] Example 3
[0033] Determination of the antioxidant capacity of the peptides obtained in Example 2:
[0034] (1) Determination of ABTS free radical scavenging rate:
[0035] The ABTS working solution was prepared by mixing ABTS (7 mmol / L) and potassium persulfate (2.45 mmol / L) at a ratio of 1:1 (v / v) and was allowed to react in the dark for 12-16 hours. The ABTS working solution was diluted with PBS buffer solution (10 mM, pH 7.4) to an absorbance of 0.70±0.02 at 734 nm, as the reaction solution. Then, 100 μL of the synthetic peptide sample (1 mg / L) and the ABTS reaction solution were added to a 96-well plate and reacted for 10 minutes, and then the absorbance was measured at 734 nm as A i . The above test was repeated with PBS instead of the sample as a blank control to determine the absorbance as A0, and each sample was repeated in 6 replicates. The ABTS free radical scavenging rate of the sample to be tested was calculated according to the following formula:
[0036]
[0037] (2) DPPH free radical scavenging rate determination:
[0038] 100 μL of the synthetic peptide sample to be tested and 100 μL of DPPH free radical solution (0.1 mmol / L) were added to a 96-well plate and placed in the dark for 30 min, and then the absorbance was measured at 517 nm and recorded as A i ; another 100 μL of the sample to be tested was added to 100 μL of absolute ethanol, and the absorbance was measured and recorded as A j ; 100 μL of 0.1 mmol / L DPPH absolute ethanol solution and 100 μL of absolute ethanol were reacted as a reference, and the absorbance was recorded as A0. Each sample was repeated in six replicates. The DPPH free radical scavenging rate of the synthetic peptide sample to be tested was calculated according to the following formula:
[0039]
[0040] (3) ·OH free radical scavenging rate determination
[0041] 2 mL of the synthetic peptide sample to be tested, 2 mL of FeSO4 (6 mmol / L) and 2 mL of H2O2 (2 mmol / L) solution were taken respectively, mixed uniformly and placed for 10 min. Then 2 mL of salicylic acid (6 mmol / L) solution was added and mixed uniformly and placed for 30 min, and the absorbance was measured at 510 nm. Each sample was independently repeated three times. The ·OH free radical scavenging rate of the synthetic peptide sample to be tested was calculated according to the following formula:
[0042]
[0043] wherein A0 refers to the absorbance value after distilled water instead of the synthetic peptide sample, A i refers to the absorbance value of the normal test group (synthetic peptide sample), A J refers to the absorbance value after distilled water instead of salicylic acid, and the rest is the same as A i group.
[0044] (4) Reducing power analysis
[0045] The reagents PBS solution (pH 6.6, 0.2 moL / L), 1% potassium ferricyanide solution (K3Fe(CN)6), 10% trichloroacetic acid (TCA) solution, and 0.1% FeCl3 solution were prepared.
[0046] In the test tube, 2 mL of the synthetic peptide sample to be tested was added, and 2 mL of PBS buffer solution (0.2 moL / L, pH 6.6) and 2 mL of 1% K3Fe(CN)6 solution were added. The test tube was placed in a 50°C water bath for 20 min, then 2 mL of 10% TCA solution was added, and after shaking, it was centrifuged at 4000 r / min for 10 min. After centrifugation, 2 mL of supernatant was taken, 2 mL of distilled water and 0.4 mL of 0.1% FeCl3 solution were added, and after shaking, it was placed in a 50°C water bath for 10 min. Finally, the absorbance value was measured at 700 nm. Repeat the above test with distilled water instead of the synthetic peptide sample as a blank control, and repeat the experiment three times independently for each sample. The reducing power is calculated by the following formula:
[0047] Total reducing power = A Q -A0
[0048] Wherein, A Q is the absorbance value of the sample group; A0 is the absorbance value of the blank control group.
[0049] The results of the antioxidant capacity detection of each synthetic peptide are shown in Table 2.
[0050] Table 2 Antioxidant capacity detection results of synthetic peptides
[0051]
[0052]
[0053] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.
Claims
1. An antioxidant peptide derived from gastrointestinal digesta of waxy corn, characterized in that: The antioxidant peptide comprises amino acid sequences DEC, IGW, GSW and at least one of SEQ ID NO.1 to SEQ ID NO.
4.
2. A glutinous corn gastrointestinal digesta, characterized in that: The invention comprises the antioxidant peptide according to claim 1.
3. The method for preparing the glutinous corn gastrointestinal digesta according to claim 2, characterized in that: The method comprises the following steps: cooking glutinous corn and then crushing it to obtain a simulated oral chew; mixing the simulated oral chew with simulated saliva, α-amylase solution and CaCl2 solution, and stirring at 37°C to obtain an oral digest; adjusting the pH of the oral digest to 3.0, and then mixing it with a pepsin solution, simulated gastric fluid and CaCl2 solution, and stirring at 37°C for 2 hours to obtain a gastric digest; mixing the gastric digest with simulated intestinal fluid and CaCl2 solution, adjusting the pH to 7.0, and then adding a trypsin solution, stirring at 37°C for 2 hours, inactivating the enzyme, and centrifuging to obtain a supernatant to obtain a gastrointestinal digest.
4. The preparation method according to claim 3, characterized in that The simulated saliva is composed of the following raw materials in parts by volume: 15.1 parts of 0.5 mol / L KCl, 3.7 parts of 0.5 mol / L KH2PO4, 6.8 parts of 1 mol / L NaHCO3, 0.5 parts of 0.15 mol / L MgCl2 and 0.06 parts of 0.5 mol / L (NH4)2CO3.
5. The preparation method according to claim 3, characterized in that The simulated gastric fluid is composed of the following raw materials in parts by volume: 6.9 parts of 0.5 mol / L KCl, 0.9 parts of 0.5 mol / L KH2PO4, 12.5 parts of 1 mol / L NaHCO3, 11.8 parts of 2 mol / L NaCl, 0.4 parts of 0.15 mol / L MgCl2 and 0.5 parts of 0.5 mol / L (NH4)2CO3.
6. The preparation method according to claim 3, characterized in that The simulated intestinal fluid is composed of the following raw materials in parts by volume: 6.8 parts of 0.5 mol / L KCl, 0.8 parts of 0.5 mol / L KH2PO4, 42.5 parts of 1 mol / L NaHCO3, 9.6 parts of 2 mol / L NaCl and 1.1 parts of 0.15 mol / L MgCl2.
7. The preparation method according to claim 3, characterized in that The concentration of the CaCl2 solution is 0.3 mol / L; the weight-to-volume ratio of the simulated oral chew to the simulated saliva, the α-amylase solution, and the CaCl2 solution is 10 g:8.95 mL:1 mL:50 μL; and the concentration of the α-amylase solution is 37 U / mL.
8. The preparation method according to claim 3, characterized in that The weight-to-volume ratio of the oral chewable material to the pepsin solution, simulated gastric fluid, and CaCl2 solution was 10 g:2 mL:17.99 mL:10 μL, and the concentration of the pepsin solution was 1800 U / mL.
9. The preparation method according to claim 3, characterized in that The weight-to-volume ratio of the oral chewable material to the simulated intestinal fluid, the CaCl2 solution, and the trypsin solution was 5 g:17.99 mL:10 μL:2 mL, and the concentration of the trypsin solution was 250 U / mL.
10. Use of the antioxidant peptide according to claim 1, the glutinous corn gastrointestinal digesta according to claim 2, or the preparation method according to any one of claims 3 to 9 in the preparation of antioxidant products.