Corn peptide-pea peptide composite peptide as well as preparation method and application thereof

Abstract

The invention relates to the technical field of food development, in particular to a corn peptide-pea peptide composite peptide and a preparation method and application thereof. The composite peptide provided by the invention consists of corn peptide and pea peptide, the mass ratio of the corn peptide to the pea peptide is (1-4): 1, and the content of Lys in the composite peptide is greater than 1.5 g / 100g protein. Compared with corn peptide, the composite peptide provided by the invention has remarkable advantages in the aspects of amino acid score, amino acid ratio coefficient, amino acid ratio coefficient score and the like; the bitterness value is obviously reduced, and the palatability is good; the compound disclosed by the invention has good anti-oxidation, anti-inflammatory and anti-apoptosis activities on a cellular level, has antagonistic and protective effects on H2O2-induced Caco-2 cell damage, can be applied to preparation of related foods, medicines and health-care products, and has good market application and popularization values.

Description

Technical Field

[0001] This invention relates to the field of food development technology, and in particular to a corn peptide-pea peptide composite peptide, its preparation method, and its application. Background Technology

[0002] The World Health Organization (WHO) reports that diet is the second leading factor affecting human health, after genetic factors. Developing health foods suitable for specific populations that are natural, safe, and highly nutritious has become a research hotspot in both the medical and food science fields. In recent years, food-derived protein hydrolysates and small peptides have received widespread attention due to their diverse biological activities, low molecular weight, easy absorption, and lack of toxic side effects. By altering protein structure, the effects of certain amino acid residues and specific sequences on oxygen free radicals can be enhanced, thereby significantly improving their antioxidant and other biological activities. Enzymatic preparation of protein hydrolysates is currently the main method for enhancing protein biological activity. Existing studies have shown that protein hydrolysates from plant sources such as corn, soybeans, wheat germ, rapeseed, and peas all possess varying degrees of biological activity, providing a material basis for precise nutrition and health regulation for specific populations.

[0003] Corn gluten meal (CGM) is a byproduct of the wet starch processing of corn, rich in zein (65–68%) and glutenin (22–33%). Zein contains approximately 50% nonpolar amino acids (such as Leu, Pro, and Ala), is insoluble in water, but soluble in aqueous ethanol, aqueous acetone, and alkaline solutions with a pH ≥ 11.5. Glutenin, on the other hand, is composed of more than 20 subunits with molecular weights of 11–127 kDa linked by disulfide bonds, exhibiting a complex structure. It is soluble only in dilute acid or alkaline solutions, and insoluble in neutral aqueous solutions and ethanol. This unique solubility characteristic limits the widespread application of corn gluten in the food industry, thus necessitating the development of high-value-added utilization pathways.

[0004] Corn peptides are low-molecular-weight products obtained from corn protein through protease hydrolysis or microbial fermentation. Compared to water-insoluble corn protein, corn peptides have significantly improved water solubility and possess various physiological functions, such as promoting alcohol metabolism, protecting the liver, anti-fatigue, lowering blood sugar, inhibiting angiotensin-converting enzyme, anti-oxidation, anti-inflammation, and anti-cancer effects. These bioactive peptides typically consist of 2-20 amino acids with a molecular weight between 300-1000 Da, making them easily digestible and absorbable, and showing broad prospects in the food, pharmaceutical, and health food industries. However, studies have found that corn peptides and corn protein have similar amino acid compositions, with lysine (Lys) being the first limiting amino acid in both, resulting in lower overall amino acid bioavailability. Furthermore, during enzymatic hydrolysis, hydrophobic amino acid residues are exposed at the peptide ends, causing the hydrolysate to have a bitter taste; the intensity of bitterness is positively correlated with the number of terminal hydrophobic groups. Reducing the degree of hydrolysis can alleviate bitterness, but it leads to decreased conversion rate and insufficient release of physiological functions. Poor palatability has, to some extent, limited the application of corn protein in the food industry.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a corn peptide-pea peptide composite peptide with balanced amino acid composition, good palatability, and multiple health functions, as well as its preparation method and applications.

[0007] Specifically, the technical solution of the present invention is as follows:

[0008] In a first aspect, the present invention provides a complex peptide composed of corn peptide and pea peptide, wherein the mass ratio of corn peptide to pea peptide is 1-4:1, and the content of Lys in the complex peptide is >1.5g / 100g protein.

[0009] Preferably, the mass ratio of corn peptides to pea peptides is as follows: 1.0:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3.0:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, or 4.0:1.

[0010] Preferably, the content of Lys in the complex peptide is 1.79-4.30g / 100g protein.

[0011] Preferably, the bitterness of the complex peptide is determined using an electronic tongue, and the determination result meets at least one of the following criteria:

[0012] The measured value of the B-bitterness2 AN0 sensor is ≤13.57;

[0013] The measured value of the Aftertaste-B C00 sensor is ≤0.97;

[0014] The measured value of the Aftertaste-A AE1 sensor is ≥ -0.56;

[0015] The measured value of the H-bitterness BT0 sensor is ≤10.85;

[0016] The measured value of the Bitterness C00 sensor is ≤20.30;

[0017] The measured value of the Astringency AE1 sensor is ≤4.62.

[0018] Preferably, the corn peptide is prepared by enzymatic hydrolysis of corn gliadin with Alcalase for 2±0.5 h at a temperature of 60±5℃ and a pH of 8.5±0.5.

[0019] More preferably, in one more specific embodiment provided by the present invention, the preparation method of the corn peptide is as follows: Corn gliadin is added to distilled water to prepare a suspension with a substrate mass concentration of 4%-10% (w / v). The suspension is placed in a 55-65℃ water bath with a magnetic stirrer. A pH meter electrode is inserted into the suspension, and the pH is adjusted to 8.0-8.5 with 1.0 mol / L NaOH. After maintaining the pH at 8.0-8.5 for 2.5-3.5 minutes, Alcalase (1-3% enzyme, enzyme to substrate mass ratio) is immediately added to begin the enzymatic hydrolysis reaction. During the enzymatic hydrolysis, 1.0 mol / L NaOH is continuously added to maintain the pH at 8.0-8.5. After 1.75-2.5 hours of enzymatic hydrolysis, the enzyme is immediately inactivated by boiling in a water bath for 10-15 minutes. After cooling to room temperature, the mixture is centrifuged at 3000-4000 r / min for 10 minutes. The supernatant is then freeze-dried for 48 hours to obtain the corn peptide.

[0020] Preferably, the pea peptide is prepared by the following method: pea protein is hydrolyzed with papain for 2±0.5 h at a temperature of 50±5℃ and a pH of 8.0±0.5.

[0021] More preferably, in one more specific embodiment provided by the present invention, the method for preparing the pea peptide is as follows: Pea protein is added to distilled water to prepare a suspension with a substrate concentration of 8%-12% (w / v). The suspension is placed in a magnetic stirrer in a water bath at 48-52℃. A pH meter electrode is inserted into the suspension, and the pH is adjusted to 8.0 with 1.0 mol / L NaOH and maintained at this pH for 3 minutes. Papain (1-3% enzyme, enzyme to substrate mass ratio) is then added, and the mixture is enzymatically hydrolyzed at 48-52℃ for 1.5-2.5 hours. During the hydrolysis process, 1.0 mol / L NaOH is continuously added dropwise to maintain the pH at 8.0. After the hydrolysis is complete, the mixture is immediately removed and inactivated by boiling in a water bath for 8-10 minutes. After cooling to room temperature, it is centrifuged at 3000-4000 r / min for 10-15 minutes. The supernatant is then freeze-dried for 48 hours to obtain the pea peptide.

[0022] Preferably, the SRC value of the complex peptide is ≥90.98.

[0023] Secondly, the present invention provides a method for preparing the aforementioned complex peptide, comprising the following steps: hydrolyzing zein with alcalase for 2±0.5 h at a temperature of 60±5℃ and a pH of 8.5±0.5, and separating the zein to obtain zein; hydrolyzing pea protein with papain for 2±0.5 h at a temperature of 50±5℃ and a pH of 8.0±0.5, and separating the pea peptide; and mixing the zein and the pea peptide at a mass ratio of 1-4:1.

[0024] Thirdly, the present invention provides the application of the aforementioned complex peptide in the preparation of products that antagonize oxidative damage to intestinal mucosal cells.

[0025] Preferably, the product has a protective effect on Caco-2 cells damaged by H2O2.

[0026] Beneficial effects:

[0027] This invention provides a corn peptide-pea peptide composite peptide, its preparation method, and its applications. The composite peptide of this invention is composed of corn peptide and pea peptide, with a mass ratio of corn peptide to pea peptide of 1-4:1, and the Lys content in the composite peptide is >1.5g / 100g protein. Compared with corn peptide, the composite peptide provided by this invention exhibits significant advantages in amino acid score, amino acid ratio coefficient, and amino acid ratio coefficient score; the bitterness value is significantly reduced, resulting in good palatability; and it possesses good antioxidant, anti-inflammatory, and anti-apoptotic activities at the cellular level, exhibiting antagonistic and protective effects against H2O2-induced damage to Caco-2 cells. It can be applied to the preparation of related foods, pharmaceuticals, and health products, and has good market application and promotion value. Detailed Implementation

[0028] To meet the precise nutrition and health regulation needs of specific populations, this invention employs a strategy of adding pea peptides rich in Lys to prepare a corn peptide-pea peptide complex peptide without compromising the health functions of corn peptides. This not only balances the amino acid composition of corn peptides and reduces bitterness, but also endows the complex peptide with multiple health functions. It improves the palatability of corn peptides while achieving the goal of precise nutrition and health regulation, and can be developed into a health product suitable for sub-healthy individuals.

[0029] This invention combines corn peptides with pea peptides rich in Lys. The amino acid score (AAS), amino acid ratio coefficient (RC), and score of RC (SRC) of the corn peptides significantly increased, while the bitterness value significantly decreased. Furthermore, it exhibited good antioxidant, anti-inflammatory, and anti-apoptotic activities at the cellular level. For example, when corn peptides and pea peptides were combined in a 2:1 ratio, the AAS and RC values ​​of Lys increased to 0.46 and 1.02, respectively, and the SRC of the composite peptide increased to 91.64. Compared with the H2O2 model group, at a concentration of 100 μg / mL, the survival rate of Caco-2 cells in the composite peptide group was 114.44 ± 1.08%, indicating that the 2:1 corn peptide and pea peptide composite peptide exhibited a protective effect against H2O2-induced damage to Caco-2 cells. Compared with the H2O2 model group, at a concentration of 25 μg / mL, intervention with the 2:1 complex of corn peptides and pea peptides reduced the levels of BAD, Bcl-2, IL-6, TNF-α, Caspase3, MDA, and NF-κB in Caco-2 cells by 20.45%, 18.50%, 14.44%, 2.23%, 12.04%, 17.79%, and 2.80%, respectively, while increasing the levels of GSH-Px, SOD, and IL-10 by 30.49%, 39.85%, and 8.93%, respectively. This indicates that the 2:1 complex of corn peptides and pea peptides inhibited H2O2-induced damage to Caco-2 cells through antioxidant, anti-inflammatory, and anti-apoptotic effects. The corn peptide and pea peptide complex provided by this invention has a balanced amino acid composition, good palatability, and antagonistic and protective effects against H2O2-induced damage to Caco-2 cells, and can be applied to the preparation of related foods, pharmaceuticals, and health products.

[0030] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are all conventional methods; the materials and reagents used are all commercially available.

[0031] Example 1

[0032] This embodiment provides a corn peptide-pea peptide complex peptide with a balanced amino acid composition, good palatability, and multiple health functions.

[0033] The corn peptide-pea peptide complex peptide provided in this embodiment is prepared by the following steps:

[0034] (1) Hydrolyze the zein suspension, centrifuge to collect the supernatant, and freeze-dry to obtain zein peptide powder. The specific steps are as follows: Add zein to distilled water to prepare a suspension with a substrate mass concentration of 10% (w / v), place it in a 60℃ water bath with a magnetic stirrer, insert the pH meter electrode into the suspension, adjust the pH to 8.5 with 1.0 mol / L NaOH, maintain the pH at 8.5 for 3 min, and immediately add Alcalase alkaline protease (3% enzyme amount, enzyme to substrate mass ratio) to start the enzymatic hydrolysis reaction. During the enzymatic hydrolysis process, continuously add 1.0 mol / L NaOH to maintain the pH at 8.5. After 2 h of enzymatic hydrolysis, immediately inactivate the enzyme in a boiling water bath for 15 min, cool to room temperature, centrifuge at 4000 r / min for 10 min, collect the supernatant and freeze-dry for 48 h to obtain zein peptide.

[0035] (2) Hydrolyze the pea protein suspension, centrifuge to collect the supernatant, and freeze-dry to obtain pea peptide powder. The specific steps are as follows: Add pea protein to distilled water to prepare a suspension with a substrate concentration of 8% (w / v), place it in a 50℃ water bath with a magnetic stirrer, insert the pH meter electrode into the suspension, adjust the pH to 8.0 with 1.0 mol / L NaOH, and maintain it unchanged for 3 min. Then add papain (1% enzyme amount, enzyme to substrate mass ratio), and enzymatically hydrolyze at 50℃ for 2 h. During the enzymatic hydrolysis, continuously add 1.0 mol / L NaOH to maintain the pH at 8.0. After the enzymatic hydrolysis is completed, immediately remove it and inactivate the enzyme in a boiling water bath for 10 min. After cooling to room temperature, centrifuge at 4000 r / min for 10 min, collect the supernatant and freeze-dry for 48 h to obtain pea peptide.

[0036] (3) Preparation of corn peptide-pea peptide complex peptide. The specific steps are as follows: corn peptide and pea peptide are thoroughly mixed at a mass ratio of 2:1 to obtain corn peptide-pea peptide complex peptide.

[0037] Example 2

[0038] This embodiment provides a corn peptide-pea peptide complex peptide, the preparation method of which includes the following steps:

[0039] (1) Hydrolyze the zein suspension, centrifuge to collect the supernatant, and freeze-dry to obtain zein peptide powder. The specific steps are as follows: Add zein to distilled water to prepare a suspension with a substrate mass concentration of 8% (w / v), place it in a 65℃ water bath with a magnetic stirrer, insert the pH meter electrode into the suspension, adjust the pH to 8.2 with 1.0 mol / L NaOH, maintain the pH at 8.2 for 2.5 min, and immediately add Alcalase alkaline protease (2% enzyme amount, enzyme to substrate mass ratio) to start the enzymatic hydrolysis reaction. During the enzymatic hydrolysis, continuously add 1.0 mol / L NaOH to maintain the pH at 8.2. After 2.5 h of enzymatic hydrolysis, immediately inactivate the enzyme in a boiling water bath for 10 min, cool to room temperature, centrifuge at 3500 r / min for 10 min, collect the supernatant and freeze-dry for 48 h to obtain zein peptide.

[0040] (2) Hydrolyze the pea protein suspension, centrifuge to collect the supernatant, and freeze-dry to obtain pea peptide powder. The specific steps are as follows: Add pea protein to distilled water to prepare a suspension with a substrate mass concentration of 10% (w / v), place it in a 48℃ water bath with a magnetic stirrer, insert the pH meter electrode into the suspension, adjust the pH to 8.0 with 1.0 mol / L NaOH, and maintain it unchanged for 3 min. Then add papain (2% enzyme amount, enzyme to substrate mass ratio), and enzymatically hydrolyze at 48℃ for 2.5 h. During the enzymatic hydrolysis, continuously add 1.0 mol / L NaOH to maintain the pH at 8.0. After the enzymatic hydrolysis is completed, immediately remove it and inactivate the enzyme in a boiling water bath for 10 min. After cooling to room temperature, centrifuge at 3000 r / min for 10 min, collect the supernatant and freeze-dry for 48 h to obtain pea peptide.

[0041] (3) Preparation of corn peptide-pea peptide complex peptide. The specific steps are as follows: corn peptide and pea peptide are thoroughly mixed in a mass ratio of 1:1 to obtain corn peptide-pea peptide complex peptide.

[0042] Example 3

[0043] This embodiment provides a corn peptide-pea peptide complex peptide, the preparation method of which includes the following steps:

[0044] (1) Hydrolyze the zein suspension, centrifuge to collect the supernatant, freeze-dry to obtain zein peptide powder. The specific steps are as follows: Add zein to distilled water to prepare a suspension with a substrate concentration of 4% (w / v), place it in a 57℃ water bath with a magnetic stirrer, insert the pH meter electrode into the suspension, adjust the pH to 8.0 with 1.0 mol / L NaOH, maintain the pH at 8.0 for 3.5 min, and immediately add Alcalase alkaline protease (1% enzyme amount, enzyme to substrate mass ratio) to start the enzymatic hydrolysis reaction. During the enzymatic hydrolysis, continuously add 1.0 mol / L NaOH to maintain the pH at 8.0. After 1.75 h of enzymatic hydrolysis, immediately inactivate the enzyme in a boiling water bath for 10 min, cool to room temperature, centrifuge at 4000 r / min for 15 min, collect the supernatant and freeze-dry for 48 h to obtain zein peptide.

[0045] (2) Hydrolyze the pea protein suspension, centrifuge to collect the supernatant, and freeze-dry to obtain pea peptide powder. The specific steps are as follows: Add pea protein to distilled water to prepare a suspension with a substrate concentration of 12% (w / v), place it in a 52℃ water bath with a magnetic stirrer, insert the pH meter electrode into the suspension, adjust the pH to 8.0 with 1.0 mol / L NaOH, and maintain it at 8.0 for 3 min. Then add papain (3% enzyme amount, enzyme to substrate mass ratio), and enzymatically hydrolyze at 52℃ for 1.5 h. During the enzymatic hydrolysis, continuously add 1.0 mol / L NaOH to maintain the pH at 8.0. After the enzymatic hydrolysis is completed, immediately remove the suspension and inactivate the enzyme in a boiling water bath for 8 min. After cooling to room temperature, centrifuge at 3500 r / min for 15 min, collect the supernatant and freeze-dry for 48 h to obtain pea peptide.

[0046] (3) Preparation of corn peptide-pea peptide complex peptide. The specific steps are as follows: corn peptide and pea peptide are thoroughly mixed at a mass ratio of 4:1 to obtain corn peptide-pea peptide complex peptide.

[0047] Example 4

[0048] In this embodiment, corn peptide powder and pea peptide powder were prepared according to the method of Example 1. Then, the prepared corn peptide powder and pea peptide powder were mixed in a mass ratio of 1:1, 2:1, 3:1 and 4:1 to obtain four corn peptide-pea peptide composite peptide products, and the following tests and analyses were performed.

[0049] (I) Amino acid composition analysis: The amino acid composition of corn peptide-pea peptide is shown in Table 1.

[0050] Table 1. Amino acid composition of corn peptide-pea peptide complex (g / 100g protein)

[0051]

[0052] Note: E / N is EAA / NEAA; E / T is EAA / total amino acids.

[0053] Table 1 shows that the corn peptide-pea peptide complex contains 17 amino acids, consistent with the amino acid composition of corn peptide. With increasing pea peptide content in the complex, the total amino acid content, E / N ratio, and E / T ratio all gradually decreased, and were all lower than those of corn peptide.

[0054] Regarding essential amino acids: as the amount of pea peptide added to the complex peptide increases, the total amount of Ile, Leu, Phe, Tyr, and Phe+Tyr, as well as the total amount of essential amino acids for the human body, gradually decreases, while the content of Lys shows a gradual increasing trend.

[0055] Regarding non-essential amino acids: with the increase of pea peptides in the complex peptides, the content of Ser, Glu (Gln), Ala, Pro, and the total amount of human non-essential amino acids gradually decreased, while the content of Arg, Asp (Asn), and Gly showed a gradual decreasing trend, and all were higher than those of corn peptides. These results indicate that the addition of pea peptides balanced the amino acid composition of corn peptides.

[0056] (ii) Amino acid score (AAS) of corn peptide-pea peptide complex: The AAS values ​​of corn peptide-pea peptide complex are shown in Table 2.

[0057] Table 2. AAS values ​​of corn peptide-pea peptide complex peptides

[0058]

[0059] Table 2 shows that the AAS value of Lys in corn peptides is 0.06, making it the first LAA of corn peptides. When the ratio of corn peptide to pea peptide is 1:1, the first LAA is Cys + Met, with an AAS value of 0.05, while the AAS value of Lys increases to 0.63. Lys is an essential amino acid for the human body, promoting human development, improving the body's calcium absorption capacity, enhancing immunity, stimulating the secretion of pepsin and gastric acid, and improving the function of central nervous system tissues. By mixing pea peptides and corn peptides to form a complex peptide, the deficiency of Lys in corn peptides can be compensated for while meeting the needs of adults.

[0060] (iii) The amino acid ratio coefficient (RC) of the corn peptide-pea peptide complex.

[0061] The amino acid ratios of the corn peptide-pea peptide complex are shown in Table 3.

[0062] Table 3. Amino acid ratios of corn peptide-pea peptide complex peptides

[0063]

[0064] If the amino acid composition of a food is the same as the pattern amino acid, then all ratio coefficients should be equal to 1. If the ratio coefficient is greater than or less than 1, it indicates a deviation from the amino acid pattern. RC>1 indicates that the amino acid is relatively abundant, RC<1 indicates that the amino acid is relatively deficient, and the amino acid with the smallest RC value is the limiting amino acid.

[0065] The RC values ​​of the corn peptide-pea peptide complex were calculated using the amino acid ratio formula, as shown in Table 4.

[0066] Table 4. Ratio coefficient (RC) of corn peptide-pea peptide complex.

[0067]

[0068] As shown in Table 4, the limiting amino acid of corn peptide is Lys, with an RC value of 0.12. After the corn peptide-pea peptide complex is formed, the amino acid composition of the complex peptide becomes more balanced with the increase of pea peptide. The content of Leu and Phe+Tyr decreases relative to the excess amino acid content, while the Lys content increases.

[0069] The more dispersed the RC values, the larger the CV values, and the smaller the SRC values, the worse the nutritional value of the protein. The greater the negative contribution of these amino acids to the amino acid balance.

[0070] The amino acid ratio coefficients of the corn peptide-pea peptide complex are shown in Table 5.

[0071] Table 5. SRC (Symptom Ratio Coefficient) of Corn Peptide-Pea Peptide Complex

[0072]

[0073] Table 5 shows that corn peptides have the lowest SRC value. With the increase of pea peptides, the SRC value of the complex peptides gradually increases. When the ratio of corn peptides to pea peptides is 1:1, the SRC value increases by 4.00%. This may be because corn peptides lack Lys, and the addition of pea peptides increases the Lys content in the complex peptides, resulting in a more balanced amino acid composition. This indicates that the addition of pea peptides significantly improves the nutritional quality of corn peptides.

[0074] (iv) Bitterness of corn peptide and corn peptide-pea peptide complex: The bitterness of corn peptide and corn peptide-pea peptide complex was determined by electronic tongue, and the results are shown in Table 6.

[0075] Table 6. Results of bitterness determination of corn peptides and corn peptide-pea peptide complex.

[0076]

[0077] Note: AN0 sensor: basic bitterness sensor; BTO sensor: responds to bitterness caused by hydrochloride salts; CO0 sensor: can test almost all bitter components in food and herbal medicines; AE1 sensor: can test for astringent taste caused by astringent substances in food and medicine.

[0078] As shown in Table 6, with the increase of pea peptide content, the bitterness value of the complex peptide gradually decreased, significantly improving the palatability of corn peptide.

[0079] (V) Study on the effect of corn peptide-pea peptide complex peptide on antagonizing oxidative damage of intestinal mucosal cells.

[0080] First, the protective effect of the complex peptide against H2O2-induced damage to Caco-2 cells was determined:

[0081] (1) Take Caco-2 cells in the logarithmic growth phase and seed them into 24-well plates. Add 1 mL of cell suspension to each well and incubate at 37°C for 24 h.

[0082] (2) The experiment was divided into a control group, an H2O2 model group and a sample group (corn peptide-pea peptide = 1-4:1). 1 mL of compound peptide solution was added to each well of the sample group to make the final sample concentration 100 µg / mL. 1 mL of DMEM medium was added to each well of the control group and the H2O2 model group and cultured at 37℃ for 2 h.

[0083] (3) Discard the liquid in the wells. Add 2 mL of DMEM medium to each well of the control group, and add 2 mL of 40 mmol / L H2O2 solution to each well of the H2O2 model group and the sample group. Incubate at 37℃ for 4 h.

[0084] (4) The cells were washed twice with 500 μL PBS solution. The survival rate of Caco-2 cells in the H2O2 model group and the sample group was determined by the MTT method. The results are shown in Table 7.

[0085] Table 7 Cell viability

[0086]

[0087] Note: Different lowercase letters in the table indicate significant differences in the analysis results (P < 0.05).

[0088] The results showed that at a concentration of 100 μg / mL, both corn peptide and pea peptide 1-4:1 complex peptides exhibited protective effects against H2O2-induced damage to Caco-2 cells, especially the corn peptide and pea peptide 2:1 complex peptide, which resulted in the highest Caco-2 cell survival rate of 114.44 ± 1.08%. Secondly, the complex peptides also affected the levels of biochemical factors in H2O2-induced damaged Caco-2 cells.

[0089] (1) Take Caco-2 cells in the logarithmic growth phase and seed them into 6-well plates. Add 2 mL of cell suspension to each well and incubate at 37°C for 24 h.

[0090] (2) The experiment was divided into a control group, an H2O2 model group and a sample group (corn peptide-pea peptide = 2:1). 2 mL of compound peptide solution was added to each well of the sample group to make the final concentration of the sample 25 µg / mL. 2 mL of DMEM medium was added to each well of the control group and the H2O2 model group and cultured at 37℃ for 2 h.

[0091] (3) Discard the liquid in the wells. Add 4 mL of DMEM medium to each well of the control group, and add 4 mL of 40 mmol / L H2O2 solution to each well of the H2O2 model group and the sample group. Incubate at 37℃ for 4 h.

[0092] (4) Wash the cells twice with 1 mL PBS solution, add trypsin to digest the cells, collect the cell suspension in a sterile centrifuge tube, centrifuge at 1000 r / min for 5 min, and discard the supernatant.

[0093] (5) Add high-efficiency RIPA lysis buffer to the cell pellet, lyse at 4°C for 30 min, centrifuge at 10000 r / min for 5 min, collect the supernatant and store it in a -80°C refrigerator for later use.

[0094] (6) Take an appropriate amount of lysis buffer and place it in an ice-water bath. Follow the steps in the kit instructions to detect the content of MDA, SOD, NF-κB, TNF-α, Bcl-2, BAD and Caspase-3 in the lysis buffer.

[0095] The experimental results are shown in Table 8.

[0096] Table 8. Effects of the corn peptide-pea peptide (2:1 ratio) complex peptide on intracellular biochemical factors in Caco-2 cells.

[0097]

[0098] Table 8 shows that, compared with the H2O2 model group, when the mass concentration was 25 μg / mL, the intervention of the 2:1 complex of corn peptide and pea peptide reduced the contents of BAD, Bcl-2, IL-6, TNF-α, Caspase3, MDA, and NF-κB in Caco-2 cells by 20.45%, 18.50%, 14.44%, 2.23%, 12.04%, 17.79%, and 2.80%, respectively, while increasing the contents of GSH-Px, SOD, and IL-10 by 30.49%, 39.85%, and 8.93%, respectively. This indicates that the 2:1 complex of corn peptide and pea peptide inhibited H2O2-induced damage to Caco-2 cells through anti-oxidation, anti-inflammation, and anti-apoptosis.

[0099] The embodiments described above are merely illustrative of several implementations of the present invention, designed to facilitate a detailed understanding of the technical solutions of the present invention. However, they should not be construed as limiting the scope of patent protection. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A complex peptide, characterized in that, The complex peptide is composed of corn peptide and pea peptide, with a mass ratio of corn peptide to pea peptide of 1-4:1, and the content of Lys in the complex peptide is >1.5g / 100g protein.

2. The complex peptide according to claim 1, characterized in that, The mass ratio of corn peptides to pea peptides is as follows: 1.0:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3.0:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1 or 4.0:

1.

3. The complex peptide according to claim 1, characterized in that, The content of Lys in the complex peptide is 1.79-4.30g / 100g protein.

4. The complex peptide according to claim 1, characterized in that, The bitterness of the complex peptide was determined using an electronic tongue, and the result met at least one of the following criteria: The measured value of the B-bitterness2 AN0 sensor is ≤13.57; The measured value of the Aftertaste-B C00 sensor is ≤0.97; The measured value of the Aftertaste-A AE1 sensor is ≥ -0.56; The measured value of the H-bitterness BT0 sensor is ≤10.85; The measured value of the Bitterness C00 sensor is ≤20.30; The measured value of the Astringency AE1 sensor is ≤4.

62.

5. The complex peptide according to claim 1, characterized in that, The corn peptide was prepared by the following method: corn gliadin was enzymatically hydrolyzed with Alcalase for 2±0.5 h at a temperature of 60±5℃ and a pH of 8.5±0.

5.

6. The complex peptide according to claim 1, characterized in that, The pea peptides were prepared by the following method: pea protein was hydrolyzed with papain for 2±0.5 h at a temperature of 50±5℃ and a pH of 8.0±0.

5.

7. The complex peptide according to claim 1, characterized in that, The SRC value of the complex peptide is ≥90.

98.

8. The method for preparing the complex peptide according to any one of claims 1-7, characterized in that, The process includes the following steps: At a temperature of 60±5℃ and a pH of 8.5±0.5, zein is enzymatically hydrolyzed using Alcalase for 2±0.5 hours, and then separated to obtain zein; at a temperature of 50±5℃ and a pH of 8.0±0.5, pea protein is enzymatically hydrolyzed using papain for 2±0.5 hours, and then separated to obtain pea peptide; the zein and pea peptide are then mixed at a mass ratio of 1-4:

1.

9. The use of the complex peptide according to any one of claims 1-7 in the preparation of products antagonizing oxidative damage to intestinal mucosal cells.

10. The application according to claim 9, characterized in that, The product has a protective effect against H2O2-induced damage to Caco-2 cells.

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