Chickpea sprout peptide as well as application and preparation method thereof

Abstract

The invention discloses a chickpea sprout peptide. The amino acid sequence of the chickpea sprout peptide is as follows: Glu-Glu-Cys-Pro-Cys-Ala-Asn-Cys-Cys, wherein the amino acid sequence of the chickpea sprout peptide is as follows: Glu-Glu-Cys-Pro-Cys-Ala-Asn-Cys-Cys; the preparation method of the chickpea sprouting peptide comprises the following steps: step 1, sprouting chickpeas until the sprouting length is 0.5-1.5 cm, wherein the sprouting time is 12-72 hours; step 2, mixing and pulping germinated chickpeas and water according to a weight ratio of 1: 1-1: 1.2, and adjusting the pH value of the pulp to 6.5-8.0; step 3, adding neutral protease and compound flavor protease, and performing enzymolysis at the enzymolysis temperature of 50-55 DEG C for 2-3 hours; step 4, treating the enzymatic hydrolysate through an ultrafiltration membrane with the molecular weight cut-off of 5000Da under the conditions that the working pressure is 0.15-0.25 MPa and the working temperature is 20-45 DEG C, and collecting filtrate; and step 5, carrying out corrosion prevention and drying on the filtrate to obtain the chickpea sprout peptide. The chickpea sprout peptide has the effects of resisting obesity, resisting oxidation and adjusting immunity, in addition, the preparation method can achieve efficient and high-quality preparation of the chickpea sprout peptide, the chickpea sprout peptide is accurately screened, and the ineffective experiment cost is reduced.

Description

Technical Field

[0001] The field of the present invention is the technical field of polypeptide preparation, and specifically relates to a chickpea sprout peptide, its application and preparation method. Background Art

[0002] Obesity has become a global health problem, and its occurrence is closely related to the dysfunction of the brain-gut-fat axis. Traditional treatment methods (such as drug intervention or surgery) have limitations such as large side effects and limited application scope. Therefore, there is an urgent need to develop a safe and effective natural active substance. Chickpea sprout peptide (CSP), as a plant-derived active ingredient, has biological activities such as antioxidant and immunomodulatory effects, but its anti-obesity function has not been systematically studied. At present, the experimental-based polypeptide screening method has problems such as low efficiency and high cost, and it is difficult to accurately target the complex physiological mechanisms related to obesity, resulting in a long development cycle and low success rate of anti-obesity active peptides. The Chinese invention patent with the publication number CN114231582A discloses a chickpea peptide, its preparation method and application. The preparation method includes reacting a chickpea protein suspension with a mixed solution of alkaline protease for a reaction time greater than 1.5 h, inactivating the enzyme after the reaction, and separating to obtain the initial chickpea peptide. The chickpea peptide obtained by the preparation method provided by the present invention has good α - glucosidase inhibitory activity, good water solubility, fast absorption, and is not likely to cause allergic reactions, and can be applied to fields such as pharmaceutical compositions, health products, and food additives. Although the preparation of α - glucosidase inhibitory peptide has been achieved, there are the following problems: (1) Using a single alkaline protease for enzymatic hydrolysis results in a low polypeptide yield; (2) The correlation between the germination state and the polypeptide composition has not been established, and the germination time control is not precise enough; (3) There is a lack of a precise screening strategy for obesity-related targets, and the disclosed separation method cannot effectively enrich anti-obesity active peptides. Summary of the Invention

[0003] The purpose of the present invention is to provide a preparation method of chickpea sprout peptide and a screening method of chickpea sprout peptide. The chickpea sprout peptide of the present invention has anti-obesity, antioxidant and immunomodulatory effects. In addition, the preparation method of the present invention can achieve the preparation of chickpea sprout peptide with high efficiency and high quality, and can accurately screen chickpea sprout peptide, reducing the cost of ineffective experiments.

[0004] The technical solution of the present invention: A chickpea sprout peptide, the amino acid sequence of the chickpea sprout peptide is as follows: Glu-Glu-Cys-Pro-Cys-Ala-Asn-Cys-Cys.

[0005] The application of the aforementioned chickpea sprout peptide, the anti-obesity chickpea sprout is used to prepare functional foods or drugs with anti-obesity, antioxidant or immunomodulatory effects.

[0006] The aforementioned application, wherein the chickpea germ peptide is used to increase the activities of antioxidant enzymes SOD, CAT, and GSH-Px and reduce the level of MDA.

[0007] The aforementioned application, wherein the chickpea germ peptide is used to increase the expression levels of cytokines IL-4, IL-6, and TNFα.

[0008] The preparation method of the aforementioned chickpea germ peptide specifically includes the following steps:

[0009] Step 1: Germinate chickpeas until the bud length is 0.5 - 1.5 cm, and the germination time is 12 - 72 hours;

[0010] Step 2: Mix the germinated chickpeas with water at a weight ratio of 1:1 - 1:1.2 to make a slurry, and adjust the pH of the slurry to 6.5 - 8.0;

[0011] Step 3: Add neutral protease and compound flavor protease, and enzymatically hydrolyze at an enzymatic hydrolysis temperature of 50 - 55°C for 2 - 3 hours;

[0012] Step 4: Treat the enzymatic hydrolysate through an ultrafiltration membrane with a molecular weight cut-off of 5000 Da under the conditions of a working pressure of 0.15 - 0.25 MPa and a working temperature of 20 - 45°C, and collect the filtrate;

[0013] Step 5: Preserve and dry the filtrate to obtain the chickpea germ peptide.

[0014] In the preparation method of the aforementioned chickpea germ peptide, in Step 1, the germination time is 36 hours.

[0015] In the preparation method of the aforementioned chickpea germ peptide, in Step 2, the weight ratio of the germinated chickpeas to water is 1:1.15, and the pH value of the slurry is 7.2.

[0016] In the preparation method of the aforementioned chickpea germ peptide, in Step 5, the preservation process is to keep the filtrate at 70 - 80°C or add ozone water with a concentration of 20 - 25 mg / L.

[0017] The screening method of the aforementioned chickpea germ peptide includes the following steps:

[0018] Step a: Construct a database containing the amino acid sequence, physicochemical properties, and bioactivity data of the chickpea germ peptide;

[0019] Step b: Integrate the biomarker data related to the brain-gut-fat axis and establish a support vector machine prediction model;

[0020] Step c: Predict the binding activities of the chickpea germ peptide to obesity-related proteins BMP-7 and CD36 through the support vector machine prediction model, and screen out the candidate sequences of the chickpea germ peptide;

[0021] Step d: Verify the binding ability of the candidate sequence to obesity-related proteins BMP-7 and CD36 through molecular docking experiments.

[0022] In the aforementioned method for preparing chickpea germ peptides, the molecular weight range of the screened chickpea germ peptide candidate sequences is 100 - 450 daltons.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The chickpea germ peptides prepared by the present invention are rich in cysteine, can form a stable spatial conformation through disulfide bonds, enhance antioxidant activity and molecular stability, lay a structural foundation for their biological activity, and the small peptides obtained by molecular weight screening are more easily absorbed by the human body. The chickpea germ peptides prepared by the present invention effectively alleviate obesity and related metabolic disorders induced by oxidative stress by increasing the activities of antioxidant enzymes (SOD, CAT, GSH-Px) and reducing the level of lipid peroxidation product (MDA), and can also significantly increase the expression levels of cytokines IL-4, IL-6 and TNFα, balance the immune homeostasis, and indirectly inhibit obesity-related chronic inflammation. In addition, the present invention significantly improves the extraction efficiency and purity of chickpea germ peptides by optimizing the germination time, enzymatic hydrolysis conditions and ultrafiltration parameters during the preparation process, while ensuring that the chickpea germ peptides have stable biological activity. The present invention can achieve the precise screening of highly binding peptides to key obesity proteins through a machine learning prediction model based on support vector machines, avoiding the blindness of traditional experiments and reducing the R & D cost. Description of the Drawings

[0025] Figure 1 Shows the morphological changes of chickpea sprouts at different germination stages of the present invention;

[0026] Figure 2 Shows the isoflavone contents of chickpeas and chickpea sprouts in different germination states of the present invention;

[0027] Figure 3 Shows the gel filtration chromatography spectrum of chickpea sprout peptides after ultrafiltration of the present invention;

[0028] Figure 4 Shows the schematic diagram of the SVM prediction model of the present invention;

[0029] Figure 5 Shows the comparison chart of the in vitro free radical scavenging ability of chickpea germ peptides of the present invention;

[0030] Figure 6 Shows the comparison chart of the antioxidant capacity of chickpea germ peptides of the present invention to enhance primary cells;

[0031] Figure 7 Shows the comparison chart of the immune function improvement of primary cells by chickpea germ peptides of the present invention. Detailed implementation mode

[0032] The present invention will be further described below in conjunction with the accompanying drawings and embodiments, but it shall not be used as a basis for limiting the present invention.

[0033] Embodiment 1: A preparation method of chickpea germ peptide specifically includes the following steps:

[0034] Step 1: Germinate chickpeas until the bud length is 0.5-1.5 cm, and the germination time is 12-72 hours;

[0035] Step 2: Mix the germinated chickpeas with water at a weight ratio of 1:1-1:1.2 to make a slurry, and adjust the pH of the slurry to 6.5-8.0; preferably, the weight ratio of germinated chickpeas to water is 1:1.15, and the pH value of the slurry is 7.2;

[0036] Step 3: Add neutral protease and compound flavor protease, and carry out enzymatic hydrolysis at an enzymatic hydrolysis temperature of 50-55 °C for 2-3 hours;

[0037] Step 4: Treat the enzymatic hydrolysate through an ultrafiltration membrane with a molecular weight cut-off of 5000 Da under the conditions of a working pressure of 0.15-0.25 MPa and a working temperature of 20-45 °C, and collect the filtrate;

[0038] Step 5: Keep the filtrate at 70-80 °C or add ozone water with a concentration of 20-25 mg / L to prevent microbial spoilage, and then carry out drying, etc. to obtain chickpea germ peptide.

[0039] Over time, the chickpea sprouts gradually developed and took shape. Chickpeas (as Figure 1 shown) and chickpea sprout samples at each time period were collected for subsequent peptide identification analysis. The contents of moisture, protein, amino acids, isoflavones, etc. in chickpeas and chickpea sprouts were measured according to the germination time. The isoflavone contents of chickpeas and chickpea sprouts in different germination states are as Figure 2 shown. The results show that the isoflavone content is the highest when the chickpea sprouts germinate for 36 h. Therefore, the chickpea sprouts germinated for 36 h are most suitable for protein hydrolysis to prepare polypeptides.

[0040] Through the peptideomics technology based on orbitrap mass spectrometry, the crude chickpea germ peptide (CSP) separated and extracted was identified on a large scale, and a primary structure library of chickpea germ peptide was constructed to carry out in-depth structure-activity relationship research by molecular simulation technology. The peptide structure identification results show that: the chickpea germ peptide with a 3 kDa filter membrane was separated into 5 components by gel chromatography: CSP1-CSP5, as Figure 3As shown in the figure. Through the molecular weight determination and analysis of CSP1 - CSP5, it was found that 89.57% of the molecular weight of CSP3 was between 130.45 and 894.77 Da, and 82.55% of which was between 126.72 and 397.54 Da.

[0041] In this example, the isoflavone content and polypeptide composition of chickpeas and chickpea sprouts at different germination times were analyzed, revealing the changing rules of water, protein, amino acids and isoflavones during the germination process. And the polypeptides in chickpea sprouts germinated for 36 h were separated by gel filtration chromatography. The research results show that chickpea sprouts germinated for 36 h are the most suitable samples for polypeptide preparation, and the small - molecule peptides they contain may have biological activities such as antioxidant and immunomodulatory effects. Future research can provide more theoretical support and technical guarantee for the development of functional foods from chickpea sprouts by further optimizing the germination conditions and polypeptide separation and purification techniques.

[0042] Based on the prepared chickpea germ peptides, the screening of chickpea germ peptides is also included. The screening method includes the following steps:

[0043] Step a: Construct a database containing the amino acid sequence, physicochemical properties and biological activity data of chickpea germ peptides;

[0044] Step b: Integrate the biomarker data related to the brain - gut - adipose axis and establish a support vector machine prediction model;

[0045] Step c: Predict the binding activities of chickpea germ peptides to obesity - related proteins BMP - 7 and CD36 through the support vector machine prediction model, and screen out the candidate sequences of chickpea germ peptides;

[0046] Step d: Verify the binding ability of the candidate sequences to obesity - related proteins BMP - 7 and CD36 through molecular docking experiments.

[0047] In this embodiment, multi-dimensional data of chickpea germ peptides (amino acid sequences, physicochemical properties, existing bioactivity experimental results, etc.) and biological information related to the brain-gut-fat axis (neurotransmitter levels, intestinal flora structure, fat metabolism-related indicators, etc.) are collected from online databases such as NCBI. Then, the characteristics of chickpea germ peptides (physicochemical properties, structural characteristics, etc.) are extracted, and the relevant characteristics of the brain-gut-fat axis function (neural regulation characteristics, intestinal flora characteristics, and fat metabolism characteristics) are integrated. In the process of feature extraction, the correlation threshold (P<0.05) between the feature and the target variable is used as the screening criterion, and the chi-square test or mutual information is used to screen key features to reduce redundant dimensions. Then, a stacked autoencoder (SAE) is used to jointly extract high-order features with a convolutional neural network (CNN) through unsupervised pre-training (greedy layer-by-layer training) and supervised fine-tuning. Based on the extracted features, a dataset for the machine learning model is established, and then preprocessing steps such as cleaning, standardization, and missing value processing are performed on the dataset. In dataset cleaning, noise data (experimental outliers) are removed through Z-score standardization or Min-Max normalization to eliminate the dimensional difference. Discrete data needs to be encoded, and text data needs to be tokenized. The neural regulation characteristics include neurotransmitter levels, the intestinal flora characteristics include flora abundance, and the fat metabolism characteristics include lipase activity. A machine learning prediction model is constructed based on support vector machines to predict the impact of chickpea germ peptides on the brain-gut-fat axis function; the existing dataset is used to train the machine learning prediction model, and the machine learning prediction model is optimized through means such as cross-validation to improve the prediction performance; an independent dataset is used to evaluate the machine learning prediction model (accuracy, recall rate, F1 score, etc.) to verify its generalization ability on new data; the results of the machine learning prediction model are interpreted and analyzed to identify potential key features. The kernel functions of the support vector machine model include linear kernel and Gaussian kernel. The linear kernel is suitable for linearly separable data; the Gaussian kernel is used to handle non-linear problems, and the γ parameter needs to be adjusted to control the complexity of the machine learning prediction model. The parameter optimization is carried out by grid search or genetic algorithm combined with cross-validation to optimize the penalty coefficient C and the kernel parameter γ, and then continuously optimize and screen the accuracy rate or regression error, so that the accuracy rate ≥ 85% and the regression error ≤ 0.1. In this embodiment, by constructing a machine learning (SVM) prediction model, as Figure 4 shown, it can effectively solve the result error caused by purposeful selection, that is, avoid the omission of the selection of high-efficiency chickpea germ peptides.

[0048] The results showed that the following high-score sequences were obtained through SVM prediction. Further research found that the chickpea germ peptide sequences screened in the early stage were also included, and it was confirmed through molecular docking experiments that they had the ability to bind efficiently with key obesity proteins (BMP-7, CD36). It was named chickpea germ peptide (hereinafter, also referred to as anti-obesity chickpea germ peptide AO-CSP), and its amino acid sequence was Glu-Glu-Cys-Pro-Cys-Ala-Asn-Cys-Cys (sequence abbreviation: E-E-C-P-C-A-N-C-C), and the amino acid residues were D RAVY I H PFAE CC G G I V E G Q G P O G I GAK F.

[0049] Therefore, the chickpea germ peptide for subsequent cell experiments was synthesized in vitro by using the method of biosynthesis.

[0050] Example 2: Based on the chickpea germ peptide screened in Example 1, an in vitro biological activity test was carried out. The screened chickpea germ peptide was diluted to 0, 50, 100, 500, 1000 mg / mL respectively as the samples to be tested, and was used for the rough determination of free radical scavenging ability (such as DPPH, ABTS + , H2O2, O 2- , FRAP) indicators. After exploring the appropriate concentration range, the concentration of the samples to be tested was set at intervals of 10 mg / mL for accurate determination. In this experiment, Trolox was used as the standard product, and by measuring the free radical scavenging ability (such as DPPH, ABTS + , H2O2, O 2- , FRAP) indicators, the chickpea germ peptide with the ability to bind to key obesity proteins was screened.

[0051] As Figure 5 shown, the results showed that: with the increase of the dose of the chickpea germ peptide, its free radical scavenging ability was gradually enhanced, and the most suitable in vitro free radical scavenging effect was achieved at a concentration of 30 mg.

[0052] Based on the screened chickpea germ peptides, in vitro cell experiments were carried out. Three-week-old C57BL / 6 mice were selected. After being sacrificed by cervical dislocation, they were soaked in 75% alcohol for 10 min. Clean brain, small intestine, and adipose tissues were obtained, placed in pre-cooled PBS, and cut into pieces about 1 mm×1 mm×1 mm. Subsequently, they were transferred to a sterile 50 mL centrifuge tube, and tissue digestive fluid (type II collagenase 1.5 g / L, bovine serum albumin 20 g / L, HEPES 1.2 g / L) was added, and then placed in a 37°C water bath shaker for digestion for 30 min. After the tissue cells were cultured to maturity, each tissue was randomly divided into 2 groups (n = 6): CON (complete culture medium) and CP (complete culture medium + CSP), and tissue cell samples were collected at 0, 24, and 48 h of culture for subsequent experimental studies. Then, by measuring the antioxidant activities (SOD, CAT, GSH-Px, MDA) and cytokines (IL-4, IL-6, TNFα) of the cell samples, the free radical scavenging ability was quantified based on the measurement results, so as to screen out the chickpea germ peptides with strong binding ability to key obesity proteins.

[0053] The in vitro cell activity experiments confirmed that the extracted primary cells (brain, small intestine, adipose) had high activities. As Figure 6 and Figure 7 shown, the experimental results showed that: compared with the cells in the CON group, the antioxidant activities (SOD, CAT, GSH-Px) of the mouse cells in the CP group (the group added with 10 μg AO-CSP) were significantly increased (P < 0.05), the MDA level was significantly decreased (P < 0.05), and the immune activation ability (IL-4, IL-6, TNFα) was significantly enhanced (P < 0.05), proving that the chickpea germ peptide has the efficacy of enhancing the antioxidant ability and immune function of primary cells.

[0054] The chickpea germ peptide prepared by the present invention is rich in cysteine, can form a stable spatial conformation through disulfide bonds, enhance the antioxidant activity and molecular stability, lay a structural foundation for its biological activity, and the small molecule peptides obtained by molecular weight screening are more easily absorbed by the human body. The chickpea germ peptide prepared by the present invention effectively alleviates obesity and related metabolic disorders induced by oxidative stress by increasing the activities of antioxidant enzymes (SOD, CAT, GSH-Px) and reducing the level of lipid peroxidation products (MDA), and can also significantly increase the expression levels of cytokines IL-4, IL-6, and TNFα, balance the immune homeostasis, and indirectly inhibit obesity-related chronic inflammation. In addition, the present invention significantly improves the extraction efficiency and purity of the chickpea germ peptide by optimizing the germination time, enzymatic hydrolysis conditions, and ultrafiltration parameters during the preparation process, while ensuring the stable biological activity of the chickpea germ peptide. The present invention realizes the precise screening of high-efficiency binding peptides to key obesity proteins through a machine learning prediction model based on support vector machines, avoids the blindness of traditional experiments, and reduces the R & D cost.

[0055] In summary, this embodiment proves that chickpea sprout peptides have strong free radical scavenging ability in vitro, and can significantly enhance the antioxidant activity and immune function of primary cells. Through the application of the support vector machine (SVM) prediction model, the present invention effectively screens out chickpea sprout peptide sequences with high biological activity, and verifies their binding ability to the key obesity proteins BMP-7 and CD36 through molecular docking. The in vitro synthesis and cell experiment research of chickpea sprout peptides further confirm their potential in obesity regulation. By optimizing the germination time, enzymatic hydrolysis conditions and ultrafiltration parameters during the preparation process, the present invention significantly improves the extraction efficiency and purity of chickpea sprout peptides, while ensuring stable biological activity of chickpea sprout peptides. Through the machine learning prediction model constructed based on the support vector machine (SVM), the present invention integrates the functional characteristics of the brain-gut-fat axis, realizes the precise screening of high-efficiency binding peptides for key obesity proteins, avoids the blindness of traditional experiments, and reduces the R & D cost. The chickpea sprout peptides of the present invention have the effects of anti-obesity, antioxidant and immune regulation. In addition, the preparation method of the present invention can achieve the preparation of chickpea sprout peptides with high efficiency and high quality, precisely screen chickpea sprout peptides, and reduce the cost of ineffective experiments.

Claims

1. A chickpea germinated peptide, characterized in that, The amino acid sequence of the chickpea sprout peptide is as follows: Glu-Glu-Cys-Pro-Cys-Ala-Asn-Cys-Cys.

2. Use of the chickpea germinated peptide according to claim 1, characterized in that: The anti-obesity chickpea sprouts are used to prepare functional foods or drugs with anti-obesity, antioxidant or immunomodulatory effects.

3. The application according to claim 2, wherein: The chickpea sprout peptide is used to increase the activities of antioxidant enzymes SOD, CAT, and GSH-Px and reduce the level of MDA.

4. The application according to claim 2, wherein: The chickpea sprout peptide is used to increase the expression levels of cytokines IL-4, IL-6, and TNFα.

5. The preparation method of the chickpea germinated peptide according to any one of claims 1-4, characterized in that, Specifically, it includes the following steps: Step 1: Germinate chickpeas until the sprout length reaches 0.5 - 1.5 cm, and the germination time is 12 - 72 hours. Step 2: Mix the germinated chickpeas and water at a weight ratio of 1:1 - 1:1.2 to make a slurry, and adjust the pH of the slurry to 6.5 - 8.

0. Step 3: Add neutral protease and compound flavor protease, and enzymatically hydrolyze at an enzymatic hydrolysis temperature of 50 - 55°C for 2 - 3 hours. Step 4: Treat the enzymatic hydrolysate through an ultrafiltration membrane with a molecular weight cut-off of 5000 Da under the conditions of a working pressure of 0.15 - 0.25 MPa and a working temperature of 20 - 45°C, and collect the filtrate. Step 5: Preserve and dry the filtrate to obtain the chickpea sprout peptide.

6. The preparation method of the chickpea germinated peptide according to claim 5, characterized in that: In Step 1, the germination time is 36 hours.

7. The preparation method of the chickpea germinated peptide according to claim 5, characterized in that: In Step 2, the weight ratio of the germinated chickpeas to water is 1:1.15, and the pH value of the slurry is 7.

2.

8. The preparation method of the chickpea germ peptide according to claim 5, characterized in that: In Step 5, the preservation process is to keep the filtrate at 70 - 80°C or add ozone water with a concentration of 20 - 25 mg / L.

9. The preparation method of the chickpea germ peptide according to claim 5, characterized in that, It also includes the screening of the chickpea sprout peptide, and the screening includes the following steps: Step a: Construct a database containing the amino acid sequence, physicochemical properties, and biological activity data of the chickpea sprout peptide. Step b: Integrate the biomarker data related to the brain-gut-fat axis and establish a support vector machine prediction model. Step c: Predict the binding activities of the chickpea sprout peptide to obesity-related proteins BMP-7 and CD36 through the support vector machine prediction model, and screen out the candidate sequences of the chickpea sprout peptide. Step d: Verify the binding ability of the candidate sequences to obesity-related proteins BMP-7 and CD36 through molecular docking experiments.

10. The preparation method of the chickpea sprout peptide according to claim 9, characterized in that: The molecular weight range of the screened candidate sequences of the chickpea sprout peptide is 100 - 450 daltons.

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