Hydrolysis method and application of oligopeptide

Abstract

The invention relates to the technical field of biology, in particular to a hydrolysis method and application of oligopeptide. According to the method, the oligopeptide with the effect of reducing cholesterol is obtained through enzymolysis separation from the waste soybean meal for the first time, the attributes of the soybean meal are fully considered in the preparation method, the oligopeptide is prepared by adopting a two-time composite enzymolysis process and combining an ultrasonic-assisted means, and compared with an existing single enzymolysis process, the oligopeptide has the advantages that the content of cholesterol in the oligopeptide is reduced; the method has outstanding dominant positions, can be used for effectively obtaining the oligopeptide with excellent cholesterol lowering activity, has a blood fat lowering effect, and has a relatively great application prospect and market.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically to a method for hydrolyzing an oligopeptide and its application. Background Art

[0002] Soybean oligopeptides are small molecule protein fragments or amino acid chains with a length of 2-10 amino acids obtained after hydrolysis, separation, purification and other operations of soybean protein. Small molecule soybean oligopeptides have low viscosity, good solubility, good water absorption, and low osmotic pressure. They are easier to digest, absorb and utilize by the body than soybean protein, and show better and more extensive biological activity than soybean protein. This makes soybean oligopeptides a research hotspot in the field of functional food, medicine and feed. As the world's largest soybean consumer, China's soybean consumption and processing are mostly concentrated in soybean pressing and traditional soy products. Among new soy products, the development of soybean oligopeptide-related products mostly remains in the laboratory stage. Therefore, there is still a large development space and market prospect for the industrial production of soybean oligopeptides in my country. Dyslipidemia is a common disease, which is an abnormal metabolism of lipoproteins in the human body, mainly including total cholesterol and low-density lipoprotein cholesterol, increased triglycerides and / or decreased high-density lipoprotein cholesterol. Dyslipidemia is one of the important factors leading to atherosclerosis and an independent risk factor for coronary heart disease and ischemic stroke. Existing treatment methods cannot effectively achieve the purpose of lowering blood lipids, and there is an urgent need to provide a new treatment method. Summary of the invention

[0003] In view of the technical problems existing in the prior art, the present invention provides a method and application of hydrolysis of oligopeptides. The present invention is the first to obtain oligopeptides with the effect of lowering cholesterol by enzymatic separation from discarded soybean meal. The preparation method fully considers the properties of soybean meal, adopts a two-step composite enzymatic hydrolysis process combined with ultrasound-assisted means to prepare the oligopeptide. Compared with the existing single enzymatic hydrolysis process, the oligopeptide has an outstanding advantage, can effectively obtain oligopeptides with superior cholesterol-lowering activity, has the effect of lowering blood lipids, and has a large application prospect and market.

[0004] Specifically, the present invention first provides a method for hydrolyzing an oligopeptide, comprising the following steps: 1) Take soybean meal and add an aqueous solution containing 2M glacial acetic acid and 2M citric acid at a material-water ratio of 1:(2-7) to wash away impurities; 2) draining the washed soybean meal from step 1), adding deionized water at a mass ratio of 1:(5-15), and beating the soybean meal to form a homogenate; 3) After filtering the homogenate of step 2), add 2M NaOH to adjust the pH to 8.5-9.5 to obtain the homogenate of step 3), which is set aside; 4) Prepare a composite enzymatic solution 1, and prepare pectinase, papain and alkaline protease according to the international enzyme activity unit 1: (3-7): (5-9) as an aqueous solution for use, and the total enzyme activity unit in each 100 mL of water solution is 90-170 IU; 5) Add the composite enzymatic hydrolysate 1 of step 4) to the homogenate of step 3) in a volume ratio of (10-20):(1-3), maintain the temperature at 55-65°C for 3.5-8.5 hours, and then quickly raise the temperature to 95°C for 10-20 minutes after the enzymatic hydrolysis; 6) Prepare the composite enzymatic solution 2, and prepare the neutral protease and β-glucosidase into an aqueous solution according to the international enzyme activity unit (2-5): (1-3) dosage ratio for use, and the total enzyme activity unit in each 100 mL of water solution is 60-160 IU; 7) Collect the enzymatic hydrolysate from step 5) and add the composite enzymatic hydrolysate 2 from step 6) in a volume ratio of (5-15):(1-7). Meanwhile, place the mixed solution in an ultrasonic instrument to assist enzymatic hydrolysis. The ultrasonic treatment conditions are: 100-150KHZ ultrasound for 5 seconds followed by a 5-second interval, and continuous treatment for 10-20 minutes. Meanwhile, the liquid temperature is maintained at no more than 40-50°C, and the enzymatic hydrolysis treatment is performed for 6.5-10 hours. 8) The enzymatic hydrolysate of step 7) is filtered by ultrafiltration membrane, and the polypeptide molecules with a molecular weight cutoff of 800-1500 Da are retained. The retained enzymatic hydrolysate is subjected to LC-MS / MS analysis to analyze its sequence characteristics; and the collected oligopeptides are concentrated by low-temperature vacuum; 9) The vacuum concentrated solution in step 8) is transferred to a spray dryer to collect dry powder.

[0005] Preferably, the material-water mass ratio in step 1) is 1:2, 1:5 or 1:7.

[0006] Preferably, in step 2), the mass ratio is 1:5, 1:10, or 1:15.

[0007] Preferably, the pH of step 3) is 8.5, 9.0 or 9.5.

[0008] Preferably, in step 4), the usage ratio of pectinase, papain and alkaline protease is 1:3:5, 1:5:7 or 1:7:9.

[0009] Preferably, in step 4), the total enzyme activity units in the composite enzymatic hydrolysate 1 are 90 IU, 130 IU, or 170 IU.

[0010] Preferably, in step 5), the volume ratio is 10:1, 15:2 or 20:3.

[0011] Preferably, in step 6), the ratio of neutral protease to β-glucosidase is 2:1, 3:2 or 5:3.

[0012] Preferably, in step 6), the total enzyme activity units in the composite enzymatic hydrolysate 2 are 60 IU, 100 IU, or 160 IU.

[0013] Preferably, in step 7), the volume ratio is 5:1, 10:3 or 15:7.

[0014] Preferably, the oligopeptide sequences separated in step 8) are shown in SEQ ID NO. 1-5.

[0015] Preferably, the oligopeptide sequences separated in step 8) are shown in SEQ ID NO. 1-3 in sequence.

[0016] Another aspect of the present invention is to provide the use of oligopeptides in the preparation of drugs having the effect of lowering cholesterol.

[0017] Preferably, the oligopeptide sequences are shown in SEQ ID NO.1-5 in sequence.

[0018] Preferably, the oligopeptide sequences are shown in SEQ ID NO.1-3 in sequence.

[0019] Another object of the present invention is to provide a medicine with the effect of lowering cholesterol, wherein the medicine uses the oligopeptide as the main active ingredient, and the amino acid sequence of the oligopeptide is shown in any one of SEQ ID NO.1-5.

[0020] Further preferably, the amino acid sequence of the hydrolyzed polypeptide is shown in any one of SEQ ID NO.1-3.

[0021] The advantages of the present invention are as follows: the present invention obtains oligopeptides with cholesterol-lowering efficacy from waste soybean meal by enzymatic separation for the first time, the preparation method fully considers the properties of soybean meal, adopts two composite enzymatic hydrolysis processes combined with ultrasound-assisted means to prepare the oligopeptides, and the oligopeptides have outstanding advantages over the existing single enzymatic hydrolysis process, can effectively obtain oligopeptides with superior cholesterol-lowering activity, have lipid-lowering efficacy, and have great application prospects and markets. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0023] The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention.

[0024] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0025] Example 1 A method for hydrolyzing an oligopeptide comprises the following steps: 1) Take soybean meal and add an aqueous solution containing 2M glacial acetic acid and 2M citric acid at a material-water ratio of 1:2 to wash and remove impurities; 2) draining the washed soybean meal from step 1), adding deionized water at a mass ratio of 1:5, and beating the soybean meal to form a homogenate; 3) After filtering the homogenate in step 2), add 2M NaOH to adjust the pH to 8.5 for later use; 4) Prepare compound enzymatic hydrolysate 1, mix pectinase, papain and alkaline protease in an amount ratio of 1:3:5 according to the international enzyme activity unit to prepare an aqueous solution for standby use, and the total enzyme activity unit in every 100 mL of water solution is 90 IU; 5) Add the composite enzymatic hydrolysate 1 of step 4) to the homogenate of step 3) at a volume ratio of 10:1, maintain the temperature at 55°C for 3.5 hours, and then quickly heat to 95°C for 10 minutes after the enzymatic hydrolysis; 6) Prepare compound enzymatic solution 2, mix neutral protease and β-glucosidase in an international enzyme activity unit ratio of 2:1 to prepare an aqueous solution for standby use, and the total enzyme activity unit in every 100 mL of water solution is 60 IU; 7) Collect the enzymatic hydrolysate from step 5) and add the composite enzymatic hydrolysate 2 from step 6) at a volume ratio of 5:1. Meanwhile, place the mixed solution in an ultrasonic instrument to assist enzymatic hydrolysis. The ultrasonic treatment conditions are 100KHZ ultrasound for 5 seconds followed by a 5-second interval, and continuous treatment for 10 minutes. Meanwhile, the liquid temperature is maintained at no more than 40°C, and the enzymatic hydrolysis treatment is performed for 6.5 hours. 8) The enzymatic hydrolysate of step 7) is filtered by ultrafiltration membrane, and the polypeptide molecules with a molecular weight cutoff of 800-1500 Da are retained. The retained enzymatic hydrolysate is subjected to LC-MS / MS analysis to analyze its sequence characteristics; and the collected oligopeptides are concentrated by low-temperature vacuum; 9) The vacuum concentrated solution in step 8) is transferred to a spray dryer to collect dry powder.

[0026] The results showed that a total of 5 oligopeptides were obtained through sequencing and identification analysis, with sequences shown in SEQ ID NO.1-5. After purity measurement, the purity of the obtained oligopeptides could reach 82.5%, 85.4%, 75.2%, 90.7% and 86.9% respectively.

[0027] Example 2 Screening of oligopeptides with cholesterol-lowering activity: The five oligopeptides obtained in Example 1 were analyzed for their binding ability to cholesterol esterase using molecular docking technology, wherein the smaller the binding ability value, the better the docking effect and the higher the potential for cholesterol-lowering activity. The details are shown in the following table: Table 1 Analysis of binding ability of oligopeptides Binding capacity / Kcal Oligomeric peptide 1 -3.26 Oligomeric peptide 2 -5.28 Oligomeric peptide 3 -1.22 Oligomeric peptide 4 2.58 Oligomeric peptide 5 6.25 As can be seen from the above table, oligopeptides 1-3 have lower binding capacity values ​​than oligopeptides 4-5, suggesting that they may have superior cholesterol-lowering activity.

[0028] Example 3 Animal experiment analysis: A total of 70 healthy adult male Wistar rats weighing 180-200g were included. Among them, 10 rats in the blank control group were fed with ordinary feed, and the rest of the rats were fed with high-fat feed. After 2 weeks of continuous feeding, the serum total cholesterol (TC), total triglyceride (TG), low-density lipoprotein (LDL-C), and high-density lipoprotein (HDL-C) levels of rats in the blank group and model group were measured. The results are shown in Table 2 below. Compared with the blank group, the serum TC, TG, and LDL-C of the model group rats were much higher than those of the blank group mice, proving that the animal model was successfully constructed.

[0029] Table 2 Analysis of TC, TG, LDL-C and HDL-C contents in rat serum Total cholesterol (TC, mmol / L) Total triglycerides (TG, mmol / L) Low-density lipoprotein (LDL-C, mmol / L) High-density lipoprotein (HDL-C, mmol / L) Blank group 1.55±0.85 0.85±0.05 1.12±0.24 1.84±0.12 Model Group 5.68±0.20 1.85±0.47 3.57±0.55 1.34±0.15 Subsequently, the model group was equally divided into 5 groups, with 10 rats in each group. Each group was fed with a high-fat diet supplemented with 5% by mass ratio of oligopeptides 1-5, while the control group was still fed with normal diet. After 2 weeks of continuous feeding, the serum TC, TG, LDL-C and HD of the rats in each group were measured.

[0030] Table 3 Analysis of TC, TG, LDL-C and HDL-C contents in rat serum Total cholesterol (TC, mmol / L) Total triglycerides (TG, mmol / L) Low-density lipoprotein (LDL-C, mmol / L) High-density lipoprotein (HDL-C, mmol / L) Model control group 5.78±0.20 1.90±0.12 3.66±0.23 1.45±0.12 Model Group 1 1.76±0.45 0.98±0.25 1.35±0.15 1.62±0.05 Model Group 2 1.65±0.34 0.96±0.42 1.26±0.24 1.75±0.08 Model Group 3 1.95±0.12 1.15±0.35 1.48±0.26 1.45±0.07 Model Group 4 3.45±0.42 1.45±0.48 1.95±0.38 1.02±0.02 Model Group 5 4.26±0.32 1.64±0.52 2.86±0.42 0.98±0.09 The results are shown in Table 3. Compared with oligopeptides 4-5, oligopeptides 1-3 have better lipid-lowering activity, especially oligopeptide 2 has the best lipid-lowering effect.

[0031] Example 4 A method for hydrolyzing an oligopeptide comprises the following steps: 1) Take soybean meal and add an aqueous solution containing 2M glacial acetic acid and 2M citric acid at a material-water mass ratio of 1:5 to wash and remove impurities; 2) draining the washed soybean meal in step 1), adding deionized water in a mass ratio of 1:10, and beating the soybean meal to form a homogenate; 3) After filtering the homogenate in step 2), add 2M NaOH to adjust the pH to 9.0 for later use; 4) Prepare composite enzymatic solution 1, mix pectinase, papain and alkaline protease in an international enzyme activity unit ratio of 1:5:7 to prepare an aqueous solution for standby use, and the total enzyme activity unit in each 100 mL of water solution is 130 IU; 5) Add the composite enzymatic hydrolysate 1 of step 4) to the homogenate of step 3) at a volume ratio of 15:2, maintain the temperature at 55°C for 6 hours, and then quickly heat to 95°C for 15 minutes after the enzymatic hydrolysis; 6) Prepare composite enzymatic solution 2, mix neutral protease and β-glucosidase in an international enzyme activity unit ratio of 3:2 to prepare an aqueous solution for standby use, and the total enzyme activity unit in every 100 mL of water solution is 100 IU; 7) Collect the enzymatic hydrolysate from step 5) and add the composite enzymatic hydrolysate 2 from step 6) at a volume ratio of 10:3. Meanwhile, place the mixed solution in an ultrasonic instrument to assist enzymatic hydrolysis. The ultrasonic treatment conditions are 120 KHZ ultrasound for 5 seconds followed by a 5-second interval, and continuous treatment for 15 minutes. Meanwhile, the liquid temperature is maintained at no more than 45° C., and the enzymatic hydrolysis treatment is performed for 8 hours. 8) The enzymatic hydrolysate of step 7) is filtered by ultrafiltration membrane, and the polypeptide molecules with a molecular weight cutoff of 800-1500 Da are retained. The retained enzymatic hydrolysate is subjected to LC-MS / MS analysis to analyze its sequence characteristics; and the collected oligopeptides are concentrated by low-temperature vacuum; 9) The vacuum concentrated solution in step 8) is transferred to a spray dryer to collect dry powder.

[0032] Example 5 A method for hydrolyzing an oligopeptide comprises the following steps: 1) Take soybean meal and add an aqueous solution containing 2M glacial acetic acid and 2M citric acid at a material-water mass ratio of 1:7 to wash and remove impurities; 2) draining the washed soybean meal in step 1), adding deionized water in a mass ratio of 1:15, and beating the soybean meal to form a homogenate; 3) After filtering the homogenate in step 2), add 2M NaOH to adjust the pH to 9.5 for later use; 4) Prepare composite enzymatic solution 1, mix pectinase, papain and alkaline protease in an international enzyme activity unit ratio of 1:7:9 to prepare an aqueous solution for standby use, and the total enzyme activity unit in each 100 mL of water solution is 170 IU; 5) Add the composite enzymatic hydrolysate 1 of step 4) to the homogenate of step 3) at a volume ratio of 20:3, maintain the temperature at 65°C for 8.5 hours, and then quickly raise the temperature to 95°C for 20 minutes after the enzymatic hydrolysis; 6) Prepare composite enzymatic solution 2, mix neutral protease and β-glucosidase in an international enzyme activity unit ratio of 5:3 to prepare an aqueous solution for standby use, and the total enzyme activity unit in every 100 mL of water solution is 160 IU; 7) Collect the enzymatic hydrolysate from step 5) and add the composite enzymatic hydrolysate 2 from step 6) at a volume ratio of 15:7. Meanwhile, place the mixed solution in an ultrasonic instrument to assist enzymatic hydrolysis. The ultrasonic treatment conditions are 150KHZ ultrasound for 5 seconds followed by a 5-second interval, and continuous treatment for 20 minutes. Meanwhile, the liquid temperature is maintained at no more than 50°C, and the enzymatic hydrolysis treatment is performed for 10 hours. 8) The enzymatic hydrolysate of step 7) is filtered by ultrafiltration membrane, and the polypeptide molecules with a molecular weight cutoff of 800-1500 Da are retained. The retained enzymatic hydrolysate is subjected to LC-MS / MS analysis to analyze its sequence characteristics; and the collected oligopeptides are concentrated by low-temperature vacuum; 9) The vacuum concentrated solution in step 8) is transferred to a spray dryer to collect dry powder.

[0033] It is necessary to point out that the above embodiments are limited to further elaboration and explanation of the technical solution of the present invention, and are not further limitations of the technical solution of the present invention. The method of the present invention is only a preferred implementation scheme, and is not used to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for hydrolyzing an oligopeptide, characterized in that: The method comprises the following steps: 1) Take soybean meal and add an aqueous solution containing 2M glacial acetic acid and 2M citric acid at a material-water ratio of 1:(2-7) to wash away impurities; 2) draining the washed soybean meal in step 1), adding deionized water in a mass ratio of 1:(5-15), and beating the soybean meal to form a homogenate; 3) After filtering the homogenate of step 2), 2M NaOH was added to adjust the pH to 8.5-9.5 to obtain the homogenate of step 3), which was set aside; 4) Prepare a composite enzymatic solution 1, and prepare pectinase, papain and alkaline protease according to the international enzyme activity unit 1: (3-7): (5-9) as an aqueous solution for use, and the total enzyme activity unit in each 100 mL of water solution is 90-170 IU; 5) Add the composite enzymatic hydrolysate 1 of step 4) to the homogenate of step 3) in a volume ratio of (10-20):(1-3), maintain the temperature at 55-65°C for 3.5-8.5 hours, and then quickly raise the temperature to 95°C for 10-20 minutes after the enzymatic hydrolysis; 6) Prepare the composite enzymatic solution 2, and prepare the neutral protease and β-glucosidase into an aqueous solution according to the international enzyme activity unit (2-5): (1-3) dosage ratio for use, and the total enzyme activity unit in each 100 mL of water solution is 60-160 IU; 7) Collect the enzymatic hydrolysate from step 5) and add the composite enzymatic hydrolysate 2 from step 6) in a volume ratio of (5-15):(1-7). Meanwhile, place the mixed solution in an ultrasonic instrument to assist enzymatic hydrolysis. The ultrasonic treatment conditions are: 100-150KHZ ultrasound for 5 seconds followed by a 5-second interval, and continuous treatment for 10-20 minutes. Meanwhile, the liquid temperature is maintained at no more than 40-50°C, and the enzymatic hydrolysis treatment is performed for 6.5-10 hours. 8) The enzymatic hydrolysate of step 7) is filtered by ultrafiltration membrane, and the polypeptide molecules with a molecular weight cutoff of 800-1500 Da are retained. The retained enzymatic hydrolysate is subjected to LC-MS / MS analysis to analyze its sequence characteristics; and the collected oligopeptides are concentrated by low-temperature vacuum; 9) The vacuum concentrated solution in step 8) is transferred to a spray dryer to collect dry powder.

2. The method according to claim 1, characterized in that Step 1) The material-water mass ratio is 1:2, 1:5 or 1:

7.

3. The method according to claim 1, characterized in that Step 2) The mass ratio is 1:5, 1:10 or 1:

15.

4. The method according to claim 1, characterized in that The pH of step 3) is 8.5, 9.0 or 9.

5.

5. The method according to claim 1, characterized in that In step 4), the dosage ratio of pectinase, papain and alkaline protease is 1:3:5, 1:5:7 or 1:7:9; the total enzyme activity units in the composite enzymatic hydrolysate 1 are 90 IU, 130 IU or 170 IU.

6. The method according to claim 1, characterized in that In step 5), the volume ratio is 10:1, 15:2 or 20:

3.

7. The method according to claim 1, characterized in that In step 6), the usage ratio of neutral protease and β-glucosidase is 2:1, 3:2 or 5:3; the total enzyme activity units in the composite enzymatic hydrolysate 2 are 60 IU, 100 IU, and 160 IU.

8. The method according to claim 1, characterized in that In step 7), the volume ratio is 5:1, 10:3 or 15:

7.

9. The method according to claim 1, wherein The oligopeptide sequences separated in step 8) are shown in SEQ ID NO. 1-5.

10. Use of oligopeptides in the preparation of drugs having the effect of lowering cholesterol, characterized in that: The oligopeptide sequences are shown in SEQ ID NO.1-5.