Hydrolysis method and application of oligopeptide

Abstract

The present invention relates to the field of biotechnology, and more specifically, to a hydrolysis method and application of oligopeptides. The present invention for the first time enzymatically hydrolyzes and isolates oligopeptides with the efficacy of reducing cholesterol from waste soybean meal. The preparation method fully considers the properties of soybean meal, and adopts a two-step composite enzymatic hydrolysis process combined with ultrasonic assistance to prepare the oligopeptides. Compared with the existing single enzymatic hydrolysis process, the oligopeptides have a prominent advantageous position, can effectively obtain oligopeptides with excellent cholesterol-reducing activity, have the efficacy of reducing blood lipids, and have great application prospects and markets.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically, to a hydrolysis method and application of oligopeptides. Background Art

[0002] Soybean oligopeptides are small molecular protein fragments or amino acid chains with 2 - 10 amino acids obtained after hydrolysis, separation, purification, etc. of soybean protein. The small molecular soybean oligopeptides have low viscosity, good solubility, good water absorption, and low osmotic pressure. They are more easily digested, absorbed, and utilized by the body than soybean protein, and exhibit more excellent and extensive biological activities than soybean protein. This makes soybean oligopeptides a research hotspot in the fields of functional foods, medicine, and feeds. As the world's largest soybean consumer country, China's soybean consumption and processing mainly focus on soybean pressing and traditional soy products. In new soy products, the development of soybean oligopeptide-related products mostly remains at the laboratory stage. Therefore, there is still great room for development and market prospects in the industrial production of soybean oligopeptides in China. Dyslipidemia is a relatively common disease, which is the abnormal metabolism of lipoproteins in the human body, mainly including elevated total cholesterol, low-density lipoprotein cholesterol, triglycerides, and / or reduced high-density lipoprotein cholesterol, etc. 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 reducing blood lipids, and there is an urgent need to provide a new treatment method. Summary of the Invention

[0003] Aiming at the technical problems existing in the prior art, the present invention provides a hydrolysis method and application of oligopeptides. The present invention first enzymatically hydrolyzes and separates oligopeptides with the effect of reducing cholesterol from waste soybean meal. This preparation method fully considers the properties of soybean meal and uses a two-step composite enzymatic hydrolysis process combined with ultrasonic assistance to prepare the oligopeptides. Compared with the existing single enzymatic hydrolysis process, this oligopeptide has a prominent advantage, can effectively obtain oligopeptides with excellent cholesterol-lowering activity, has the effect of reducing blood lipids, and has great application prospects and markets.

[0004] Specifically, the present invention first provides a hydrolysis method of oligopeptides, including the following steps:

[0005] 1) Take soybean meal, and add an aqueous solution containing 2M glacial acetic acid and 2M citric acid according to a material-to-water mass ratio of 1:(2 - 7) for washing to remove impurities;

[0006] 2) After draining the soybean meal washed in step 1), add deionized water with a mass ratio of 1:(5 - 15), and perform pulping treatment to form a homogeneous slurry;

[0007] 3) After filtering the homogenate in step 2), adjust the pH to 8.5 - 9.5 with 2M NaOH to obtain the homogenate in step 3) for standby;

[0008] 4) Prepare the composite enzyme solution 1. Dissolve pectinase, papain, and alkaline protease in water according to the dosage ratio of international enzyme activity units 1: (3 - 7): (5 - 9) for standby. The total enzyme activity units dissolved in every 100 mL of water is 90 - 170 IU;

[0009] 5) Add the composite enzyme solution 1 in step 4) to the homogenate in step 3), and the volume ratio of the two is (10 - 20): (1 - 3). Maintain the temperature at 55 - 65 °C for enzymatic hydrolysis for 3.5 - 8.5 h, and then quickly raise the temperature to 95 °C for treatment for 10 - 20 min;

[0010] 6) Prepare the composite enzyme solution 2. Dissolve neutral protease and β - glucosidase in water according to the dosage ratio of international enzyme activity units (2 - 5): (1 - 3) for standby. The total enzyme activity units dissolved in every 100 mL of water is 60 - 160 IU;

[0011] 7) Collect the enzymatic hydrolysate in step 5), and add the composite enzyme solution 2 in step 6). The volume ratio of the two is (5 - 15): (1 - 7). At the same time, place the mixed solution in an ultrasonic instrument for assisted enzymatic hydrolysis. The ultrasonic treatment conditions are ultrasonic treatment at a frequency of 100 - 150 KHZ for 5 s and then interval for 5 s, continuously treating for 10 - 20 min; at the same time, maintain the liquid temperature not higher than 40 - 50 °C, and perform enzymatic hydrolysis treatment for 6.5 - 10 h;

[0012] 8) Filter the enzymatic hydrolysate in step 7) with an ultrafiltration membrane to retain polypeptide molecules with a molecular weight cut - off of 800 - 1500 Da. Analyze the sequence characteristics of the retained enzymatic hydrolysate by LC - MS / MS; and perform low - temperature vacuum concentration on the collected oligopeptides;

[0013] 9) Transfer the vacuum - concentrated solution in step 8) to a spray dryer for treatment to collect the dry powder.

[0014] Preferably, the mass ratio of material to water in step 1) is 1:2, 1:5, or 1:7.

[0015] Preferably, the mass ratio in step 2) is 1:5, 1:10, 1:15.

[0016] Preferably, the pH in step 3) is 8.5, 9.0, or 9.5.

[0017] Preferably, the dosage ratio of pectinase, papain, and alkaline protease in step 4) is 1:3:5, 1:5:7, or 1:7:9.

[0018] Preferably, the total enzyme activity unit in the complex enzyme hydrolysis solution 1 in step 4) is 90 IU, 130 IU, or 170 IU.

[0019] Preferably, the volume ratio in step 5) is 10:1, 15:2, or 20:3.

[0020] Preferably, the dosage ratio of neutral protease and β-glucosidase in step 6) is 2:1, 3:2, or 5:3.

[0021] Preferably, the total enzyme activity unit in the complex enzyme hydrolysis solution 2 in step 6) is 60 IU, 100 IU, or 160 IU.

[0022] Preferably, the volume ratio in step 7) is 5:1, 10:3, or 15:7.

[0023] Preferably, the oligopeptide sequences obtained by separation in step 8) are successively as shown in SEQ ID NO.1-5.

[0024] Preferably, the oligopeptide sequences obtained by separation in step 8) are successively as shown in SEQ ID NO.1-3.

[0025] Another aspect of the present invention is to provide the use of oligopeptides in the preparation of a drug with the efficacy of reducing cholesterol.

[0026] Preferably, the oligopeptide sequences are successively as shown in SEQ ID NO.1-5.

[0027] Preferably, the oligopeptide sequences are successively as shown in SEQ ID NO.1-3.

[0028] Another object of the present invention is to provide a drug with the efficacy of reducing cholesterol, the drug taking the oligopeptide as the main active ingredient, and the amino acid sequence of the oligopeptide being as shown in any one of SEQ ID NO.1-5.

[0029] Further preferably, the amino acid sequence of the hydrolyzed polypeptide is as shown in any one of SEQ ID NO.1-3.

[0030] The advantages of the present invention are as follows: The present invention first enzymatically hydrolyzes and separates oligopeptides with the efficacy of reducing cholesterol from waste soybean meal. The preparation method fully considers the properties of soybean meal and uses a two-step complex enzymatic hydrolysis process combined with ultrasonic assistance to prepare the oligopeptides. Compared with the existing single enzymatic hydrolysis process, the oligopeptides have a prominent advantageous position, can effectively obtain oligopeptides with excellent cholesterol-lowering activity, have the efficacy of reducing blood lipid, and have great application prospects and markets. Detailed implementation manners

[0031] The present invention will be further described in detail below in conjunction with specific embodiments, so that those skilled in the art can understand the present invention more clearly.

[0032] 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 those of ordinary skill in the art without creative work belong to the protection scope of the present invention.

[0033] In the embodiments 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 embodiments of the present invention, unless specifically specified, the technical means used are all conventional means well-known to those skilled in the art.

[0034] Example 1

[0035] A method for hydrolyzing oligopeptides, comprising the following steps:

[0036] 1) Take soybean meal, and add an aqueous solution containing 2M glacial acetic acid and 2M citric acid according to a material-to-water mass ratio of 1:2 for washing to remove impurities;

[0037] 2) After draining the soybean meal washed in step 1), add deionized water with a mass ratio of 1:5 and perform pulping treatment to form a homogeneous slurry;

[0038] 3) After filtering the homogeneous slurry in step 2), add 2M NaOH to adjust the pH to 8.5 for standby;

[0039] 4) Prepare a composite enzyme solution 1, and configure pectinase, papain and alkaline protease into an aqueous solution for standby according to an international enzyme activity unit dosage ratio of 1:3:5. The total enzyme activity unit in every 100 mL of water dissolution is 90 IU;

[0040] 5) Add the composite enzyme solution 1 in step 4) to the homogeneous slurry in step 3), and the volume ratio of the two is 10:1. Maintain the temperature at 55 °C for enzymatic hydrolysis for 3.5 h, and quickly raise the temperature to 95 °C after enzymatic hydrolysis for 10 min;

[0041] 6) Prepare a composite enzyme solution 2, and configure neutral protease and β-glucosidase into an aqueous solution for standby according to an international enzyme activity unit dosage ratio of 2:1. The total enzyme activity unit in every 100 mL of water dissolution is 60 IU;

[0042] 7) Collect the enzymatic hydrolysate in step 5), and add the composite enzyme solution 2 in step 6). The volume ratio of the two is 5:1. At the same time, place the mixed solution in an ultrasonic instrument for assisted enzymatic hydrolysis. The ultrasonic treatment conditions are ultrasonic for 5 s at a frequency of 100 KHZ and then interval for 5 s, and continuously treat for 10 min; at the same time, maintain the liquid temperature not higher than 40 °C and perform enzymatic hydrolysis treatment for 6.5 h;

[0043] 8) Filter the enzymatic hydrolysate in step 7) using an ultrafiltration membrane to retain polypeptide molecules with a molecular weight cut-off of 800 - 1500 Da. Analyze the sequence characteristics of the retained enzymatic hydrolysate by LC-MS / MS; and perform low-temperature vacuum concentration on the collected oligopeptides.

[0044] 9) Transfer the vacuum concentrate from step 8) to a spray dryer for treatment to collect the dry powder.

[0045] The results showed that a total of 5 oligopeptides were obtained through sequencing and identification analysis, with the sequences shown as SEQ ID NO.1 - 5. After purity determination, the purities of the obtained oligopeptides could reach 82.5%, 85.4%, 75.2%, 90.7%, and 86.9% in sequence.

[0046] Example 2

[0047] Screening of cholesterol-lowering active oligopeptides: Analyze the binding ability of the 5 oligopeptides obtained in Example 1 to cholesterol esterase using molecular docking technology. The smaller the binding ability value, the better the docking effect and the higher the potential for cholesterol-lowering activity. The specific results are shown in the following table:

[0048] Table 1 Analysis of the binding ability of oligopeptides

[0049] Binding ability / Kcal Oligopeptide 1 -3.26 Oligopeptide 2 -5.28 Oligopeptide 3 -1.22 Oligopeptide 4 2.58 Oligopeptide 5 6.25

[0050] As can be seen from the above table, oligopeptides 1 - 3 have lower binding ability values compared to oligopeptides 4 - 5, indicating that they may have superior cholesterol-lowering activity.

[0051] Example 3

[0052] Animal experiment analysis:

[0053] A total of 70 healthy adult male Wistar rats, weighing 180 - 200 g. Among them, 10 rats were in the blank control group and were fed with ordinary feed, while the remaining rats were all fed with high-fat feed. After continuous feeding for 2 weeks, the serum total cholesterol (TC), total triglyceride (TG), low-density lipoprotein (LDL-C), and high-density lipoprotein (HDL-C) levels of the rats in the blank group and the 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 rats in the model group were much higher than those of the blank group mice, proving that the animal model was successfully constructed.

[0054] Table 2 Analysis of the contents of TC, TG, LDL-C, and HDL-C in rat serum

[0055] Total cholesterol (TC, mmol / L) Total triglyceride (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

[0056] Subsequently, the model group was evenly divided into 5 groups, with 10 rats in each group. Each group was fed with a high-fat diet supplemented with 5% by mass of oligopeptides 1-5 in sequence, and the control group was still fed with a normal diet. After continuous feeding for 2 weeks, the serum TC, TG, LDL-C, and HD of rats in each group were measured.

[0057] Table 3 Analysis of the Contents of Serum TC, TG, LDL-C, and HDL-C in Rats

[0058] Total cholesterol (TC, mmol / L) Total triglyceride (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

[0059] As shown in Table 3, compared with oligopeptides 4-5, oligopeptides 1-3 have better hypolipidemic activity, especially oligopeptide 2 has the best hypolipidemic effect.

[0060] Example 4

[0061] A hydrolysis method of oligopeptides includes the following steps:

[0062] 1) Take soybean meal, add an aqueous solution containing 2M glacial acetic acid and 2M citric acid according to a solid-liquid mass ratio of 1:5 for washing to remove impurities;

[0063] 2) After draining the soybean meal washed in step 1), add deionized water with a mass ratio of 1:10 and perform pulping treatment to form a homogeneous slurry;

[0064] 3) After filtering the homogeneous slurry in step 2), add 2M NaOH to adjust the pH to 9.0 for standby;

[0065] 4) Prepare a compound enzyme solution 1, and configure pectinase, papain, and alkaline protease into an aqueous solution for standby according to an enzyme activity unit ratio of 1:5:7. The total enzyme activity unit per 100 mL of water dissolution is 130 IU;

[0066] 5) Add the compound enzyme solution 1 in step 4) to the homogeneous slurry in step 3), and the volume ratio of the two is 15:2. Maintain the temperature at 55°C for enzymatic hydrolysis for 6 h, and quickly raise the temperature to 95°C for treatment for 15 min after enzymatic hydrolysis;

[0067] 6) Prepare a compound enzyme solution 2, and configure neutral protease and β-glucosidase into an aqueous solution for standby according to an enzyme activity unit ratio of 3:2. The total enzyme activity unit per 100 mL of water dissolution is 100 IU;

[0068] 7) Collect the enzyme solution in step 5), and add the compound enzyme solution 2 in step 6). The volume ratio of the two is 10:3. At the same time, place the mixed solution in an ultrasonic instrument for assisted enzymatic hydrolysis. The ultrasonic treatment conditions are ultrasonic treatment at a frequency of 120 KHZ for 5 s and then interval for 5 s, and continuous treatment for 15 min; at the same time, maintain the liquid temperature not higher than 45°C and perform enzymatic hydrolysis treatment for 8 h;

[0069] 8) Filter the enzymatic hydrolysate in step 7) with an ultrafiltration membrane to retain polypeptide molecules with a molecular weight cut-off of 800 - 1500 Da. Analyze the sequence characteristics of the retained enzymatic hydrolysate by LC-MS / MS; and perform low-temperature vacuum concentration on the collected oligopeptides.

[0070] 9) Transfer the vacuum concentrate in step 8) to a spray dryer for treatment to collect the dry powder.

[0071] Example 5

[0072] A hydrolysis method for oligopeptides, comprising the following steps:

[0073] 1) Take soybean meal, add an aqueous solution containing 2M glacial acetic acid and 2M citric acid according to a material-to-water mass ratio of 1:7 for washing to remove impurities.

[0074] 2) After draining the washed soybean meal in step 1), add deionized water with a mass ratio of 1:15 and perform pulping treatment to form a homogeneous slurry.

[0075] 3) After filtering the homogeneous slurry in step 2), add 2M NaOH to adjust the pH to 9.5 for standby.

[0076] 4) Prepare a composite enzymatic hydrolysate 1 by mixing pectinase, papain, and alkaline protease according to an international enzyme activity unit ratio of 1:7:9 to form an aqueous solution for standby. The total enzyme activity unit in every 100 mL of water dissolution is 170 IU.

[0077] 5) Add the composite enzymatic hydrolysate 1 in step 4) to the homogeneous slurry in step 3) at a volume ratio of 20:3. Maintain the temperature at 65°C for enzymatic hydrolysis for 8.5 h, and then quickly raise the temperature to 95°C for treatment for 20 min.

[0078] 6) Prepare a composite enzymatic hydrolysate 2 by mixing neutral protease and β-glucosidase according to an international enzyme activity unit ratio of 5:3 to form an aqueous solution for standby. The total enzyme activity unit in every 100 mL of water dissolution is 160 IU.

[0079] 7) Collect the enzymatic hydrolysate in step 5) and add the composite enzymatic hydrolysate 2 in step 6) at a volume ratio of 15:7. At the same time, place the mixture in an ultrasonic instrument for assisted enzymatic hydrolysis, where the ultrasonic treatment conditions are 5 s of ultrasonic wave at a frequency of 150 KHZ followed by a 5 s interval, and continuous treatment for 20 min; meanwhile, maintain the liquid temperature not higher than 50°C and perform enzymatic hydrolysis treatment for 10 h.

[0080] 8) Filter the enzymatic hydrolysate in step 7) with an ultrafiltration membrane to retain polypeptide molecules with a molecular weight cut-off of 800 - 1500 Da. Analyze the sequence characteristics of the retained enzymatic hydrolysate by LC-MS / MS; and perform low-temperature vacuum concentration on the collected oligopeptides.

[0081] 9) Transfer the vacuum concentrated solution from step 8) to a spray dryer for treatment to collect the dry powder.

[0082] It is necessary to point out here that the above embodiments are only for further elaboration and illustration of the technical solution of the present invention, rather than a further limitation on the technical solution of the present invention. The method of the present invention is only a preferred implementation solution 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 within the protection scope of the present invention.

Claims

1. A hydrolysis method of oligopeptide, characterized in that, The method includes the following steps: 1) Take soybean meal, and add an aqueous solution containing 2M glacial acetic acid and 2M citric acid according to a material-to-water mass ratio of 1:(2 - 7) for washing to remove impurities; 2) After draining the soybean meal washed in step 1), add deionized water with a mass ratio of 1:(5 - 15), and perform pulping treatment to form a homogeneous slurry; 3) After filtering the homogeneous slurry in step 2), add 2M NaOH to adjust the pH to 8.5 - 9.5 to obtain the homogeneous slurry in step 3) for standby; 4) Prepare composite enzyme solution 1, and configure pectinase, papain, and alkaline protease into an aqueous solution for standby according to an international enzyme activity unit ratio of 1:(3 - 7):(5 - 9). The total enzyme activity unit dissolved in every 100 mL of water is 90 - 170 IU; 5) Add the composite enzyme solution 1 in step 4) to the homogeneous slurry in step 3), with a volume ratio of (10 - 20):(1 - 3). Maintain the temperature at 55 - 65°C for enzymatic hydrolysis for 3.5 - 8.5 h, and quickly raise the temperature to 95°C after enzymatic hydrolysis for 10 - 20 min; 6) Prepare composite enzyme solution 2, and configure neutral protease and β-glucosidase into an aqueous solution for standby according to an international enzyme activity unit ratio of (2 - 5):(1 - 3). The total enzyme activity unit dissolved in every 100 mL of water is 60 - 160 IU; 7) Collect the enzymatic hydrolysate in step 5), and add the composite enzyme solution 2 in step 6), with a volume ratio of (5 - 15):(1 - 7). Meanwhile, place the mixed solution in an ultrasonic instrument for assisted enzymatic hydrolysis. The ultrasonic treatment conditions are ultrasonic for 5 s at a frequency of 100 - 150 KHZ and then interval for 5 s, and continuously treat for 10 - 20 min; meanwhile, maintain the liquid temperature not higher than 40 - 50°C, and perform enzymatic hydrolysis treatment for 6.5 - 10 h; 8) Filter the enzymatic hydrolysate in step 7) with an ultrafiltration membrane to retain polypeptide molecules with a molecular weight cut-off of 800 - 1500 Da. Analyze the sequence characteristics of the retained enzymatic hydrolysate by LC-MS / MS; and perform low-temperature vacuum concentration on the collected oligopeptides; 9) Transfer the vacuum concentrate in step 8) to a spray dryer for treatment to collect dry powder; Among them, the oligopeptide sequences separated in step 8) are successively shown as SEQ ID NO.1 - 3.

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

7.

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

15.

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

5.

5. The method according to claim 1, wherein 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 unit in composite enzyme solution 1 is 90 IU, 130 IU, or 170 IU.

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

3.

7. The method according to claim 1, wherein In step 6), the dosage ratio of neutral protease and β-glucosidase is 2:1, 3:2, or 5:3; the total enzyme activity unit in composite enzyme solution 2 is 60 IU, 100 IU, or 160 IU.

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

7.

9. Use of oligopeptides in the preparation of a medicament having the efficacy of reducing cholesterol, characterized in that, The oligopeptide sequence is any one of those shown as SEQ ID NO.1 - 3.