Enzymatic hydrolysate of red jujube protein and preparation method and application thereof

Abstract

The application discloses an enzymatic hydrolysate of red date protein, which contains ACE inhibitory peptides and DPPIV inhibitory peptides; the amino acid sequence of the ACE inhibitory peptides is Arg-Leu-Pro-His-Val or Thr-Val-Lys-Pro-Gly-Leu or Lys-Ala-Leu-Val-Ala-Pro; and the amino acid sequence of the DPPIV inhibitory peptides is Tyr-Leu-His-Leu or Ile-Pro-Ile-Tyr or Lys-Ala-Leu-Val-Ala-Pro. The application further discloses a preparation method of the enzymatic hydrolysate of red date protein and application thereof. The proteinase is added in the red date protein at 0.3-2% (w / w) in terms of mass percentage, and enzymolysis is carried out at 37 DEG C for 2-3 hours, and then the enzymatic hydrolysate of red date protein is obtained through freeze-drying; or the enzymatic hydrolysate of red date protein is obtained through enzymolysis of the red date protein in the gastrointestinal tract. The enzymatic hydrolysate of red date protein has the effects of assisting in reducing blood pressure and reducing blood sugar, and can be used for preparing functional food or health-care food with the functions of assisting in reducing blood pressure and reducing blood sugar, and is particularly suitable for patients with diabetes accompanied by high blood pressure, and has a good market application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of food and medicine technology, and in particular to an enzymatic product of jujube protein and a preparation method and application thereof. BACKGROUND

[0002] Due to the fast pace of life and irregular life of people in today's society, combined with less exercise, it is easy to cause the increase of blood lipid and blood sugar in human body, leading to the increase of the incidence of hypertension and diabetes, and these two diseases have become two common diseases in today's society. Foreign relevant data shows that the prevalence rate of hypertension among diabetes patients is 40% to 80%; the report in China is lower than that abroad, which is 25% to 48.1%. During the onset period, patients need to take some auxiliary antihypertensive and hypoglycemic drugs regularly. At present, the commonly used drugs are western medicines, but these drugs often have different degrees of toxic and side effects, especially have a great impact on kidney function, and the curative effect is short. Therefore, it is of great significance to extract natural and safe antihypertensive and hypoglycemic drugs from food, and food raw materials with both antihypertensive and hypoglycemic functions will have a broader application market.

[0003] Bioactive peptides are a general term of peptides with various human metabolic and physiological regulation functions, are easily digested and absorbed, and have very high food safety, and have become a hot research topic in current food and medicine. There are many types of food-derived bioactive peptides, including angiotension converting enzyme (ACE) inhibitory peptides, dipeptidyl peptidase 4 (DPPIV) inhibitory peptides, immunomodulatory peptides, antioxidant peptides, antibacterial peptides, antithrombotic peptides, and calcium absorption promoting peptides, etc. Among them, ACE inhibitory peptides and DPPIV inhibitory peptides are closely related to the prevention and treatment of hypertension and hyperglycemia diseases.

[0004] Jujube is a traditional oriental medicine, which is rich in protein (3-7% by dry weight), carbohydrates (including 2-9% of fiber polysaccharides by dry weight), vitamins (especially vitamins B and C), and important minerals (such as manganese, iron, phosphorus, potassium, magnesium, calcium, zinc, etc.). It is reported that jujube polysaccharides have various activities, such as immunomodulation, antioxidant, and anti-inflammatory. Jujube extract has anti-allergic activity, inhibits food-borne pathogenic bacteria, regulates plasmin activity, antioxidant activity, and antibacterial activity. However, there are few reports on the use of jujube protein and its hydrolysate, and the research on other functions of jujube protein is also lacking. SUMMARY

[0005] The technical problem solved by the present application is to provide an enzymatic hydrolysate of red jujube protein with ACE and DPPIV inhibitory effects, a preparation method thereof and application.

[0006] The technical solution adopted by the present application to solve the above technical problem is:

[0007] An enzymatic hydrolysate of red jujube protein, which contains ACE inhibitory peptides and DPPIV inhibitory peptides.

[0008] An enzymatic hydrolysate of red jujube protein, wherein the amino acid sequence of the ACE inhibitory peptide is Arg-Leu-Pro-His-Val or Thr-Val-Lys-Pro-Gly-Leu or Lys-Ala-Leu-Val-Ala-Pro.

[0009] An enzymatic hydrolysate of red jujube protein, wherein the amino acid sequence of the DPPIV inhibitory peptide is Tyr-Leu-His-Leu or Ile-Pro-Ile-Tyr or Lys-Ala-Leu-Val-Ala-Pro.

[0010] A preparation method of an enzymatic hydrolysate of red jujube protein, wherein 0.3-2% (w / w) of protease is added to red jujube protein, and the red jujube protein is enzymatically hydrolyzed at 37 DEG C for 2-3 hours, and then is freeze-dried to obtain the enzymatic hydrolysate of red jujube protein, wherein the protease is selected from the group consisting of trypsin: pepsin with a mass ratio of 1:1, papain: bromelain with a mass ratio of 1:1, papain, and bromelain.

[0011] A preparation method of an enzymatic hydrolysate of red jujube protein, wherein the enzymatic hydrolysate of red jujube protein is obtained by enzymatic hydrolysis of red jujube protein in the gastrointestinal tract.

[0012] A preparation method of an enzymatic hydrolysate of red jujube protein, wherein the red jujube protein is prepared by the following method:

[0013] (1) red dates are weighed, and distilled water is added according to a solid-liquid ratio of 1:5-10, and the red dates are cooked at 110 DEG C-121 DEG C under high-pressure conditions with a set pressure value of 20-100 Kpa for 10-15 minutes, and then are peeled and cored, and are homogenized to obtain high-pressure red date juice;

[0014] (2) Take the high-pressure jujube juice prepared in step (1), add cellulase and hemicellulase for mixed enzymatic hydrolysis at a temperature of 55℃-65℃ and pH 4.5-5.5, where the amount of cellulase and hemicellulase is 0.1% of the jujube mass, the hydrolysis time is 3-6h, the enzyme is inactivated, and after cooling, add pectinase at a temperature of 50℃-60℃ and pH 3.0-4.0 for enzymatic hydrolysis at a time of 3-6h, the enzyme is inactivated, and after cooling, jujube protein hydrolysate is obtained.

[0015] (3) Take the jujube protein hydrolysate prepared in step (2), centrifuge and precipitate it, take the precipitate and dialyze it in a 500 Da dialysis bag, freeze dry it, and obtain the jujube protein.

[0016] Application of an enzymatic digestion product of jujube protein in the preparation of health products and drugs for assisting in lowering blood pressure.

[0017] Application of an enzymatic digestion product of jujube protein in the preparation of adjuvant hypoglycemic health products and adjuvant hypoglycemic drugs.

[0018] Compared with the prior art, the advantages of the present invention are as follows:

[0019] This invention optimizes the extraction process of jujube protein components and simulates enzymatic digestion of jujube protein with high content. It was found that the resulting jujube protease digestion product simultaneously exhibits ACE and DPPIV inhibitory effects. Furthermore, in vitro and in vivo experiments verified that the jujube protease digestion product simultaneously targets ACE and DPPIV for dual-target inhibition. The ACE IC50 of the jujube protease digestion product was [not specified in the original text]. 50 The concentration was 0.34-0.58 mg / mL, DPPVIC 50 With a concentration of 1.83-3.22 mg / mL and high activity, compared to most currently available enzymatic digestion products that only have one ACE inhibition function or DPPIV inhibition function, the enzymatic digestion product of this invention can simultaneously exert auxiliary effects in lowering blood pressure and blood sugar. It has a significant advantage in the preparation of functional foods or health foods with auxiliary functions in lowering blood pressure and blood sugar, and is particularly suitable for patients with diabetes and hypertension, with good market application prospects.

[0020] This invention collects reported ACE peptide and DPPIV composition datasets and training sets, creates a 3D-QSAR model, and uses the 3D-QSAR model to predict peptides with ACE and DPPIV inhibitory activities. ACE and DPPIV inhibitory peptides are synthesized and validated, yielding ACE-active inhibitory peptide sequences of Arg-Leu-Pro-His-Val, Thr-Val-Lys-Pro-Gly-Leu, and Lys-Ala-Leu-Val-Ala-Pro, IC50 The values ​​were 6.01 μM, 3.81 μM, and 17.06 μM, respectively; the DPPIV activity-inhibiting peptide sequences were Tyr-Leu-His-Leu, Ile-Pro-Ile-Tyr, and Lys-Ala-Leu-Val-Ala-Pro, with IC50 values ​​of 6.01 μM, 3.81 μM, and 17.06 μM, respectively; 50 The values ​​were 381 uM, 43.5 uM, and 360 uM, respectively; the obtained ACE inhibitory peptide and DPPIV peptide have novel structures, strong activity, and high safety. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to the embodiments.

[0022] To further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims of the present invention.

[0023] Experimental Example 1

[0024] Preparation of jujube protein

[0025] 1.1 High-Pressure Enzymatic Hydrolysis Method: Take 200g of raw material, add 2000ml of water, and cook for 15 minutes at 110℃ and a pressure of 80Kpa. Remove the skin and pits, and homogenize. First, add 0.2g of cellulase and 0.2g of hemicellulase for enzymatic hydrolysis at pH=5, T=55℃, and t=3h. Then, use 0.2g of pectinase for enzymatic hydrolysis at pH=3.5, T=50℃, and t=3h. After each enzymatic hydrolysis, the enzymes need to be inactivated at 95℃ for 15 minutes to obtain crude jujube protein extract.

[0026] 1.2 High-Pressure Method: Take 200g of raw material, add 2000ml of water, and cook for 15 minutes at 110℃ and a pressure of 80Kpa. Remove the skin and pits, and homogenize. Then, enzymatically hydrolyze the mixture using 0.2g of pectinase at pH=3.5, T=50℃, and t=3h. After each enzymatic hydrolysis, the enzyme needs to be inactivated at 95℃ for 15 minutes to obtain crude jujube protein extract.

[0027] 1.3 Room temperature method: Take 200g of raw material, remove the pits, add 2000ml of water, soak for 120 minutes, homogenize, and then enzymatically hydrolyze with 0.2g of pectinase at pH=3.5, T=50℃, t=3h. After each enzymatic hydrolysis, the enzyme needs to be inactivated at 95℃ for 15 minutes to obtain crude jujube protein extract.

[0028] Take the three crude extracts of jujube protein prepared by the above method, adjust the pH to 12, dissolve the protein in the sample, centrifuge the sample at 4000 r / min for 10 min, take the supernatant, and adjust the pH to 3.

[0029] After standing for a period of time, the protein is precipitated, centrifuged at 8000 r / min for 15 min, the precipitate is taken into a dialysis bag with a molecular weight of 500 Da, dialysis treatment is performed overnight, and the red jujube protein is obtained by freeze-drying;

[0030] The protein content in the supernatant of the dialysis bag treated by the three methods is roughly measured by BCA method, and Table 1 is shown below.

[0031] Sample name Absorbance Concentration (mg / g) High pressure enzymatic method 1.7711 23.465 High pressure method 0.688 13.785 Ambient method 0.624 12.502

[0032] Table 1: Protein content of red jujube protein in the supernatant treated by the three methods in Example 1 measured by BCA method

[0033] The experimental results show that the protein concentration value measured by high-pressure enzymolysis method is 23.465 mg / g. From the results, it can be seen that the protein content of red jujube obtained by high-pressure enzymolysis method is relatively high compared with high-pressure method and normal temperature method, and therefore the high-pressure enzymolysis method is the best method for extracting red jujube protein.

[0034] Experimental Example 2

[0035] (1) 200 g of raw material is taken, 2000 ml of water is added, and high-pressure conditions of 110°C and a set pressure value of 90 Kpa are used for cooking for 15 minutes. The skin and core are removed, and homogenization is performed. Enzymolysis conditions are as follows: 0.2 g of cellulase and 0.2 g of hemicellulase are added for enzymolysis, pH = 5, T = 55°C, and t = 3 h. Then, 0.2 g of pectinase is used for enzymolysis, pH = 3.5, T = 50°C, and t = 3 h. Enzyme inactivation is required after each enzymolysis, 95°C for 15 minutes, to obtain red jujube protein crude extract;

[0036] (2) The red jujube protein enzymolysis product prepared in the above step (1) is adjusted to pH 12, the protein in the sample is dissolved, the sample is centrifuged at 4000 r / min for 10 min, the supernatant is taken, and the pH is adjusted to 3.

[0037] (3) After standing for a period of time, the protein is precipitated, centrifuged at 8000 r / min for 15 min, the precipitate is taken into a dialysis bag with a molecular weight of 500 Da, dialysis treatment is performed overnight, and the red jujube protein is obtained by freeze-drying;

[0038] (4) The red jujube protein prepared in the above step (3) is analyzed by proteomics technology, and the parameters of the 15 kinds of proteins with the highest content are listed in Table 2. From Table 2, the Exp.q-value of the 15 kinds of proteins is 0, indicating that the results are very reliable, the peptide coverage is relatively high, the number of amino acids of the protein is from 366 to 1020, and the molecular weight range is extensive.

[0039]

[0040]

[0041] Table 2. The 15 highest content proteins in jujube protein of Example 2

[0042] Example 3

[0043] Gastrointestinal tract simulation digestion experiment of jujube protein

[0044] Take the 15 proteins with higher content in the jujube protein prepared in step (3) of Example 2 to perform a gastrointestinal tract virtual digestion experiment. The virtual digestion uses the website (https: / / biochemia.uwm.edu.pl / biopep-uwm / ). The number of active peptides generated is listed and counted. It is found that jujube protein will generate a large number of peptides with strong ACE inhibitory activity under the action of trypsin, pepsin, papain and bromelain. The in vitro virtual digestion activity table of jujube protein is listed. As can be seen from Table 3, the active peptides ranking higher have the functions of DPPIV inhibition, ACE inhibition, antioxidant, DPP-III inhibition, glucose uptake stimulation and renin inhibitor. Among them, the DPPIV inhibition and ACE inhibition inhibition activities are very strong, being 48.49% and 34.94% respectively. Therefore, the ACE and DPPIV inhibitory peptides of jujube protein are further studied through experiments.

[0045] Sequence Function Peptide segment function as a percentage of total peptide segment 1 DPPIV inhibition 48.49 2 ACE inhibition 34.94 3 Antioxidant 4.92 4 DPP-III inhibition 4.62 5 Glucose uptake stimulation 3.61 6 Renin inhibitor 3.41

[0046] Table 3. In vitro virtual digestion activity table of jujube protein

[0047] Example 4

[0048] Preparation of jujube proteinase digestion products

[0049] Take the jujube protein prepared in step (3) of Example 2, and add 0.3% (w / w) protein for enzyme hydrolysis, respectively. The enzyme hydrolysis conditions are as follows:

[0050] a) Trypsin + pepsin (w / w = 1:1)

[0051] b) Papain + bromelain (w / w = 1:1)

[0052] c) Papain

[0053] d) Bromelain

[0054] 37 degrees for 2 hours of enzyme hydrolysis, and the enzyme hydrolysate is freeze-dried to obtain jujube enzyme hydrolysate, respectively.

[0055]

[0056]

[0057] Table 4 ACE and DPPIV inhibitory activities of jujube enzymatic hydrolysates obtained by different enzymatic methods

[0058] Experimental conclusion: jujube enzymatic hydrolysates obtained by several enzymatic methods all have good ACE and DPPIV inhibitory activities. Among in vitro enzymatic methods, the combination of papain and bromelain has better activity, ACE IC 50 is 0.35 mg / ml, and DPPIV IC 50 is 1.83 mg / ml. Jujube protein mouse intestinal contents is a peptide obtained by in vivo enzymatic hydrolysis, ACE IC 50 is 0.21 mg / ml, and DPPIV IC 50 is 1.88 mg / ml.

[0059] Example 5

[0060] In vivo digestion experiment of jujube protein

[0061] Take the jujube protein prepared in step (3) of Example 2. The mice were fasted overnight before the experiment. The mice were divided into a blank group and a jujube protein group, with 3 mice in each group. The jujube protein group was administered jujube protein at a dose of 200 mg / kg by gavage. After 60 min, the mice were sacrificed, and the small intestine was removed and placed in a culture dish containing physiological saline. The outer wall was gently washed, and then the small intestine was divided into multiple sections. One milliliter of physiological saline was injected into the small intestine, and the intestinal juice and intestinal contents were gently squeezed and placed in a 2-milliliter centrifuge tube. Centrifugation was performed at 4°C and 12000 r / min for 20 min. The sample was freeze-dried to obtain jujube protein in vivo enzymatic hydrolysate. Ultra-high performance liquid chromatography (UPLC) combined with orbitrap Exactive mass spectrometer was used to identify the peptide sequences.

[0062] The red jujube mouse intestinal content sample was dissolved in 10 μL of mobile phase A (0.1% formic acid solution), and the peptides were captured on a column (PepMap C18, 100 μm x 2 cm, 5 μm) at a flow rate of 5 uL / min. After 3 min, the peptides were separated by gradient elution on a sodium analysis column chromatography (PepMap C18, 75 μm x 25 cm, 1.7 μm). The separation gradient was from 5% to 30% mobile phase B (acetonitrile) for 95 min 0.1% formic acid solution). The chromatography flow rate was 300 nL / min, and the column temperature was 45°C. The ion source spray voltage was 2.0 kV, and the QExative mass spectrometer capillary was set to 300°C. Data were collected using a data-dependent mode to automatically switch between MS and MS / MS. Full scan MS used Orbitrap to perform the main scan: the scan range was set to m / z 350-1600, and the resolution was set to 70000 (m / z 200). The data were analyzed using PeakStudio, and 118 red jujube in vivo enzymatic peptides were obtained.

[0063] Example 6

[0064] ACE and DPPIV peptide screening and verification experiment of red jujube protein

[0065] 6.1 Take the red jujube protein prepared in step (3) of Example 2, and fast the mice overnight before the experiment. Divide the mice into a blank group and a red jujube protein group, with 3 mice in each group. The red jujube protein group is given red jujube protein by gavage at a dose of 200 mg / kg. 60 min later, the mice are sacrificed, and the small intestine is removed and placed in a culture dish containing physiological saline. The outer wall is gently washed, and then the small intestine is divided into multiple sections. One milliliter of physiological saline is injected into the small intestine, and the intestinal juice and small intestine contents are gently squeezed into a 2-milliliter centrifuge tube. Centrifugation is performed at 12,000 r / min for 20 min at 4°C. The sample is freeze-dried, desalted, and subjected to ultra-high performance liquid chromatography (UPLC) combined with Orbitrap Exactive mass spectrometry to identify the peptide sequences. Peptidomic analysis shows that there are nearly 1000 peptides with ALC%>80 in the intestinal samples of mice given red jujube protein. The 705 common peptides contained in the 3 samples are used for the next experiment;

[0066] The 705 common peptides are removed from the peptides present in the intestinal tract of the blank mice, and finally 113 peptides are used for ACE and DPPIV inhibition activity screening.

[0067] 6.2 Take the peptides obtained by activity screening in step 6.1 above. Create a 3D-QSAR model by collecting the currently reported ACE and DPPIV peptide dataset and training set, and use the 3D-QSAR model to predict the ACE and DPPIV activity of the peptides prepared in step 6.1.

[0068] 6.3 The peptide fragments prepared in step 6.2 above were subjected to synthetic verification of ACE activity. The ACE inhibitory activity was determined according to the method of Sangsawad et. al (2020) with slight modifications. The polypeptides were dissolved in distilled water at a concentration of 1 mg / mL. After the reaction was terminated with 1 M HCL, the content of hippuric acid (HA) was determined by RP-HPLC with a C18 column connected to an Agilent 1260 high-performance liquid system. Elution was performed with 70% mobile phase A (pure water containing 1% TFA) and 30% mobile phase B (ACN containing 0.1% TFA) for 10 min. The retention time of hippuric acid was 4.1 min, and HHL eluted at 6.5 min. The ability of the hydrolysate to inhibit ACE activity was expressed as IC50. All analyses were performed in 5 different concentrations, with 3 replicates. The inhibition rate was calculated as follows:

[0069] Inhibition activity (%) = HA sample - HA control / HA control - HA blank x 100%

[0070] Where HA control, HA sample, and HA blank represent the relative area of the HA peak

[0071] 6.3 Dipeptidyl peptidase 4 (DPP4) inhibitor screening kit, Merck

[0072] Enzyme: diluted 50 times

[0073] Positive drug: diluted 1000 times

[0074] Substrate: diluted 12.5 times

[0075] Sample: (25 μl sample + 75 μl Buffer) pipette 25 μl + 50 μl enzyme + 25 μl substrate

[0076] Conditions: incubate at 37°C for 10 min

[0077]

[0078] Calculation formula:

[0079] Inhibition rate calculation formula = slope calculation (control - sample) / control

[0080]

[0081] Table 5 ACE inhibitory peptides identified in the mouse intestinal contents of jujube protein

[0082] The experimental conclusion is that three peptide segments with higher inhibitory activity are obtained by verifying the ACE inhibitory peptide segments obtained by virtual screening, Arg-Leu-Pro-His-Val, Thr-Val-Lys-Pro-Gly-Leu, and Lys-Ala-Leu-Val-Ala-Pro, and the IC 50 The digestion stability is higher, and the three peptide segments have greater research value and antihypertensive application value.

[0083]

[0084] Table 6 DPP-IV inhibitory peptide segments identified in the intestinal contents of mice fed with jujube protein

[0085] The experimental conclusion is that three peptide segments with higher inhibitory activity are obtained by verifying the DPP IV inhibitory peptide segments obtained by virtual screening, Tyr-Leu-His-Leu, Ile-Pro-Ile-Tyr, and Lys-Ala-Leu-Val-Ala-Pro, and the IC50 is 381 uM, 43.5 uM, and 360 uM, respectively, and the digestion stability is higher, and the three peptide segments have greater research value and antidiabetic application value.

Claims

1. An enzymatic digestion product of jujube protein, characterized in that, The amino acid sequence of the enzyme digestion product is Lys-Ala-Leu-Val-Ala-Pro.

2. The method for preparing the enzymatic digestion product of jujube protein according to claim 1, characterized in that, The jujube protein was digested by enzymes in the gastrointestinal tract to obtain the jujube protein digestion product. The jujube protein mentioned above is prepared by the following method: (1) Weigh the red dates, add distilled water at a ratio of 1:5-10, cook at 110℃-121℃ and a pressure of 20-100Kpa for 10-15 minutes, peel, remove the pits, homogenize, and obtain high-pressure red date juice. (2) Take the high-pressure jujube juice prepared in step (1), add cellulase and hemicellulase for mixed enzymatic hydrolysis at a temperature of 55℃-65℃ and pH 4.5-5.5, where the amount of cellulase and hemicellulase is 0.1% of the jujube mass, the hydrolysis time is 3-6h, the enzyme is inactivated, and after cooling, add pectinase at a temperature of 50℃-60℃ and pH 3.0-4.0 for enzymatic hydrolysis at a time of 3-6h, the enzyme is inactivated, and after cooling, jujube protein hydrolysate is obtained. (3) Take the jujube protein hydrolysate prepared in step (2), centrifuge and precipitate it, take the precipitate and dialyze it in a 500 Da dialysis bag, freeze dry it, and obtain the jujube protein.

3. The application of the enzymatic digestion product of jujube protein according to claim 1 in the preparation of health products and drugs for assisting in lowering blood pressure.

4. The application of the enzymatic digestion product of jujube protein according to claim 1 in the preparation of auxiliary hypoglycemic health products and auxiliary hypoglycemic drugs.

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