Pig blood hypoglycemic peptide as well as preparation method and application thereof

Abstract

The invention relates to the technical field of peptide extraction, in particular to a pig blood hypoglycemic peptide as well as a preparation method and application thereof. The preparation method comprises the following steps: freezing and thawing pig blood, carrying out pig blood denaturation treatment, carrying out two-step enzymolysis, and purifying to obtain the pig blood glucose-reducing peptide. The two-step enzymolysis comprises the following steps: firstly adding alkaline protease into the pig blood solution with the enzyme addition amount of 8000-10000 U / g, reacting for 3-6 hours under the conditions that the pH is 7.5-9.0 and the temperature is 45-55 DEG C, performing enzyme deactivation and cooling, then adjusting the pH to 6-7, then adding compound protease with the enzyme addition amount of 3000-5000 U / g, reacting for 1-4 hours under the condition that the temperature is 45-55 DEG C, performing enzyme deactivation and cooling, and adjusting the pH to 4.5-5.0 to obtain a compound enzymatic hydrolysate A; the purification treatment comprises the step of sequentially performing ultrafiltration by using ultrafiltration membranes of 10KDa, 5KDa and 3KDa. The pig blood glucose-reducing peptide prepared by the preparation method disclosed by the invention is high in yield and good in inhibition rate on alpha-glucosidase and alpha-amylase.

Description

Technical Field

[0001] The invention relates to the technical field of peptide extraction, and in particular to a pig blood blood glucose-lowering peptide and a preparation method and application thereof. Background Art

[0002] Diabetes is a disease that seriously endangers human life and health. Traditional treatment methods mainly include the use of hypoglycemic drugs, but these drugs may cause adverse reactions such as hypoglycemia and weight gain, and have certain toxic and side effects on the human body. In recent years, food-derived peptides have attracted widespread attention as natural hypoglycemic drugs. Plant-derived peptides such as bitter melon peptides, soybean peptides and oat peptides have been proven to have hypoglycemic effects. However, although there are some studies on hypoglycemic peptides, there are generally problems such as unsatisfactory flavor, low yield, and weak hypoglycemic function.

[0003] Pigs are the world's largest meat-producing livestock. A pig produces about 1.5-3L of blood, accounting for about 13% of the total by-products of pork production. It is often used as food raw materials, animal feed or fertilizer, with low output value and easy to cause environmental pollution. Pig blood is a nutritious food that contains a variety of nutrients beneficial to the human body. It is rich in nutrients such as vitamin B2, vitamin C, protein, iron, phosphorus, calcium and niacin. These ingredients play an important role in maintaining normal physiological functions of the human body and promoting health. Moreover, pig blood is not only high in protein, but also contains a variety of essential amino acids. It is a complete protein. Therefore, deep processing of high-quality protein in pig blood can give pig blood a higher value and improve the production efficiency of enterprises. Summary of the invention

[0004] In view of the technical problems of hypoglycemic peptides in the prior art, such as unsatisfactory flavor, low yield and weak blood sugar lowering function, the present invention provides a pig blood hypoglycemic peptide and a preparation method and application thereof. The hypoglycemic peptide is prepared using pig blood as a raw material for the first time, and the metallic astringency in the pig blood raw material is removed by purification treatment. The pig blood hypoglycemic peptide prepared by two-step enzymatic hydrolysis has a high yield and good inhibitory effect on α-glucosidase and α-amylase.

[0005] In a first aspect, the present invention provides a method for preparing a porcine blood hypoglycemic peptide, wherein the porcine blood hypoglycemic peptide is prepared through freezing and thawing of porcine blood, denaturation treatment of porcine blood, two-step enzymolysis and purification treatment; Among them, the two-step enzymatic hydrolysis is to first add alkaline protease in the pig blood solution, the enzyme amount is 8000-10000U / g, the enzymatic hydrolysis reaction is carried out for 3-6 hours under the conditions of pH 7.5-9.0 and temperature 45-55°C, the pH is adjusted to 6-7 after the enzyme is inactivated and cooled, and then the composite protease is added in an amount of 3000-5000U / g, the enzymatic hydrolysis reaction is carried out for 1-4 hours under the conditions of temperature 45-55°C, the enzyme is inactivated and cooled, and the pH is adjusted to 4.5-5.0 to obtain composite enzymatic hydrolyzate A; the purification treatment includes centrifugation, filtration and ultrafiltration using 10KDa, 5KDa and 3KDa ultrafiltration membranes in sequence.

[0006] Furthermore, the pig blood freeze-thaw process includes adding an anticoagulant to fresh pig blood, freezing at -40°C overnight, thawing at room temperature, and repeating the freeze-thaw process three times.

[0007] Furthermore, the pig blood denaturation treatment is to mix the pig blood obtained after freezing and thawing the pig blood and water in a mass ratio of 1:3 and then place the mixture in a water bath at 90-95° C. for 30-60 minutes to obtain a pig blood solution.

[0008] Furthermore, alkaline protease is subjected to enzymatic hydrolysis at a pH of 7.5-8.5 and a temperature of 50° C. After the enzymatic hydrolysis is completed, the product is boiled for 10 minutes to inactivate the enzyme, and the pH is adjusted to 7 after cooling.

[0009] Furthermore, the composite protease includes endoproteinase, exoproteinase and flavor enzyme.

[0010] Furthermore, the composite protease is subjected to enzymatic hydrolysis at a temperature of 50° C. for 1-4 hours. After the enzymatic hydrolysis is completed, the mixture is boiled for 10 minutes to inactivate the enzyme, and the pH value is adjusted to 4.5 after cooling.

[0011] Further, the purification treatment is to take the supernatant after centrifugation of the composite enzymatic hydrolysate A, adjust the pH value to 7.0, and then filter it using a 0.25μm filter membrane. The filtrate is ultrafiltered in sequence using 10KDa, 5KDa and 3KDa ultrafiltration membranes at 4°C to obtain a composite enzymatic hydrolysate B. The composite enzymatic hydrolysate B is vacuum-freeze-dried to obtain pig blood hypoglycemic peptide.

[0012] Furthermore, the centrifugal speed is 6000-10000 r / min, and the centrifugal time is 30-60 min.

[0013] In a second aspect, the present invention provides a porcine blood hypoglycemic peptide prepared by the above-mentioned preparation method.

[0014] In a third aspect, the present invention provides an application of a pig blood hypoglycemic peptide in the preparation of medicines or health foods that help maintain healthy blood sugar levels.

[0015] The beneficial effects of the present invention are: The present invention provides a method for preparing a hypoglycemic peptide from pig blood, which has a simple process and is easy to operate. The hypoglycemic peptide from pig blood obtained by two-step enzymolysis has a high yield, a good inhibition rate on α-glucosidase and α-amylase, and a high hypoglycemic activity. The present invention discovers for the first time that the hypoglycemic peptide from pig blood has potential hypoglycemic activity, which gives a higher economic value to the byproduct of pig blood. In addition, ultrafiltration using 10KDa, 5KDa and 3KDa ultrafiltration membranes in sequence during the purification process can not only prevent the ultrafiltration membrane from being blocked by macromolecular proteins and extend the service life of the ultrafiltration membrane, but also the color of the purified hypoglycemic peptide from pig blood changes from blood red to light yellow, removes the metallic astringency, improves the flavor of the product, and has broad application prospects and market potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 This is a photo of the composite enzymatic solution A after only centrifugation purification.

[0018] Figure 2 This is a photo of compound enzymatic solution B. DETAILED DESCRIPTION

[0019] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0020] A method for preparing a porcine blood hypoglycemic peptide comprises the following steps: (1) Freezing and thawing of pig blood: Add anticoagulant (0.9% sodium citrate) to fresh pig blood and freeze it at -40°C overnight. Then thaw it at room temperature. Repeat the freezing and thawing process three times to ensure that the red blood cells are completely ruptured.

[0021] (2) Pig blood denaturation treatment: The pig blood with completely ruptured red blood cells in step (1) and water are mixed in a mass ratio of 1:3 to obtain a pig blood solution. After being stirred evenly, the mixture is bathed in water at 90-95°C for 30-60 min (except for Example 6, which is bathed in water at 95°C for 60 min, the other examples and comparative examples are bathed in water at 90°C for 30 min) to obtain a pig blood solution. The pig blood protein is denatured, resulting in the pig blood solution being dark reddish brown.

[0022] (3) Two-step enzymatic hydrolysis: add alkaline protease (manufacturer: Shanghai MacLean Biochemical Technology Co., Ltd., product number: P824138-100g) to the pig blood solution, the enzyme amount is 8000-10000U / g, and the enzymatic hydrolysis reaction is carried out at pH 7.5-9.0 (pH is adjusted using 0.1mol / L sodium hydroxide and 0.1mol / L dilute hydrochloric acid) and temperature 45-55℃ for 3-6h. After the enzymatic hydrolysis is completed, boil for 10min to inactivate the enzyme, adjust the pH to 6-7 after cooling, and then perform compound protease enzymatic hydrolysis; The added enzyme amount of the composite protease (manufacturer: Shanghai Yuanye Biotechnology Co., Ltd., product number S10155-100g, mainly composed of protein endo-nuclease, protein exo-nuclease and flavor enzyme, etc.) is 3000-5000U / g. The enzymatic hydrolysis reaction is carried out at a temperature of 45-55°C for 1-4h. After the enzymatic hydrolysis is completed, the solution is boiled for 10min to inactivate the enzyme. After cooling, the pH is adjusted to 4.5-5.0 (pH is adjusted using 0.1mol / L dilute hydrochloric acid) to obtain composite enzymatic hydrolyzate A.

[0023] (4) Purification treatment: The composite enzymatic hydrolysate A was centrifuged at a speed of 6000-10000 r / min for 30-60 min (except Example 10 which was centrifuged for 30 min, the other examples and comparative examples were all centrifuged for 60 min). After centrifugation, the supernatant was taken and the pH was adjusted to 7.0 (the pH was adjusted using 0.1 mol / L sodium hydroxide). The supernatant was then filtered using a 0.25 μm filter membrane. The filtrate was ultrafiltered at 4°C using 10 KDa, 5 KDa and 3 KDa ultrafiltration membranes in sequence. The composite enzymatic hydrolysate B obtained after ultrafiltration was vacuum freeze-dried at -40°C for 36-48 h (except Example 13 which was vacuum freeze-dried for 48 h, the other examples and comparative examples were all vacuum freeze-dried for 36 h) to obtain the porcine blood hypoglycemic peptide (stored at -20°C).

[0024] The specific parameter conditions of the two-step enzymatic hydrolysis in the embodiments and comparative examples are shown in Tables 1 and 2, the calculation method of the yield of porcine blood hypoglycemic peptide is shown in Experimental Example 1, the determination method of the inhibition rate of porcine blood hypoglycemic peptide on α-glucosidase is shown in Experimental Example 2, and the determination method of the inhibition rate of porcine blood hypoglycemic peptide on α-amylase is shown in Experimental Example 3. Their results are shown in Table 3.

[0025] When the inventor prepared the porcine blood hypoglycemic peptide, he found that the color of the solution obtained by centrifuging the composite enzymatic hydrolysate A without filtration and ultrafiltration was blood red (e.g. Figure 1 ), centrifuged and filtered, and then passed through 10KDa and 5KDa ultrafiltration membranes in sequence, the obtained composite enzymatic hydrolysate B was light yellow (such as Figure 2). If only ultrafiltration is performed, large molecular proteins will block the ultrafiltration membrane with small pores. Using 10KDa, 5KDa and 3KDa ultrafiltration membranes in sequence can reduce the damage to the ultrafiltration membrane and extend the service life of the ultrafiltration membrane.

[0026] Table 1 Condition parameters for adding alkaline protease in step (3)

[0027] Table 2 Condition parameters for adding compound protease in step (3)

[0028] Experimental Example 1 Yield of Pig Blood Hypoglycemic Peptide Take 1 mL of the sample to be tested (complex enzymatic hydrolysate B) and mix with 1 mL of 10% (w / v) trichloroacetic acid solution and shake well to remove the macromolecular protein in the sample to be tested. After standing for 10 minutes, centrifuge at 6000 rpm for 10 minutes. Take 1 mL of the supernatant and mix with 4 mL of biuret reagent. Let it stand for 60 minutes and measure its absorbance at 540 nm. Substitute it into the standard curve (y=0.0509x+0.0079; R 2 =0.999) to obtain the concentration of hypoglycemic peptide in pig blood, and the yield of hypoglycemic peptide in pig blood was calculated according to the following formula:

[0029] Experimental Example 2 Determination of the Inhibition Rate of α-Glucosidase by Pig Blood Hypoglycemic Peptide Take the sample (complex enzymatic solution B) and 1 mL of 10 U / mL α-glucosidase in a PE tube, mix well, incubate at 37°C for 10 min, add 0.5 mL of 4 mmol / L pNP-G solution, mix well, and place in a 37°C incubator for 15 min. Finally, add 1 mL of anhydrous methanol to stop the reaction, and measure the absorbance of the sample group at a wavelength of 405 nm.

[0030] The blank group was phosphate buffer solution (pH 6.8) instead of the sample, the background group was phosphate buffer solution (pH 6.8) instead of α-glucosidase solution, and acarbose (concentration of 2 mg / mL) was used as the positive control. The calculation formula is as follows:

[0031] Experimental Example 3 Determination of the Inhibition Rate of α-amylase by Pig Blood Hypoglycemic Peptide Prepare 100mL of 1% (w / w) starch solution, the solvent is 0.2mol / L phosphate buffer (pH 6.8), place the starch solution in a 95℃ water bath for 8min to denature it, wait until the temperature drops to room temperature, mix well and set aside. Use a pipette to draw 20μL of sample (complex enzymatic solution B) and 10μL of α-amylase solution (1U / mL) into a test tube, mix well, and react at 37℃ for 15min. Add 500μL of pretreated starch solution and react at 37℃ for 5min. After the reaction, heat in a 100℃ water bath for 15min to terminate the reaction. After the reaction, cool to room temperature and measure the absorbance at 540nm.

[0032] The control group was added with 20 μL of phosphate buffer and 10 μL of α-amylase solution, the background group was added with 20 μL of sample (compound enzymatic solution B) and 10 μL of phosphate buffer, the positive control group was added with 20 L of 2 mg / mL acarbose solution and 10 μL of α-amylase solution, and the rest of the operation was the same as above. The calculation formula is as follows:

[0033] Table 3 Results of various embodiments and comparative examples

[0034] In addition, the composite enzymatic hydrolysate A of Example 1 was centrifuged and divided into two equal parts, one of which was ultrafiltered in sequence using 10KDa, 5KDa and 3KDa ultrafiltration membranes at 4°C, and the other was decolorized using an activated carbon decolorization process (activated carbon dosage 2.0%, pH 4.0, temperature 50°C, decolorization time 1.5h) to compare the loss rate of the porcine blood hypoglycemic peptide. The porcine blood hypoglycemic peptide obtained by the activated carbon decolorization process lost 0.14% more porcine blood hypoglycemic peptide than that obtained by ultrafiltration, and the α-glucosidase inhibition rate of the porcine blood hypoglycemic peptide obtained by the activated carbon decolorization process was 45.21±0.98%, and the α-amylase inhibition activity was 26.98±2.11%, and the activity effects were not ideal.

[0035] Although the present invention has been described in detail with reference to the accompanying drawings and in combination with the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection of the present invention.

Claims

1. A method for preparing a porcine blood hypoglycemic peptide, characterized in that: The porcine blood hypoglycemic peptide was prepared by freezing and thawing the porcine blood, denaturing the porcine blood, performing two-step enzymatic hydrolysis and purifying the porcine blood. Among them, the two-step enzymatic hydrolysis is to first add alkaline protease in the pig blood solution, the enzyme amount is 8000-10000U / g, the enzymatic hydrolysis reaction is carried out for 3-6 hours under the conditions of pH 7.5-9.0 and temperature 45-55°C, the pH is adjusted to 6-7 after the enzyme is inactivated and cooled, and then the composite protease is added in an amount of 3000-5000U / g, the enzymatic hydrolysis reaction is carried out for 1-4 hours under the conditions of temperature 45-55°C, the enzyme is inactivated and cooled, and the pH is adjusted to 4.5-5.0 to obtain composite enzymatic hydrolyzate A; the purification treatment includes centrifugation, filtration and ultrafiltration using 10KDa, 5KDa and 3KDa ultrafiltration membranes in sequence.

2. The method for preparing a porcine blood hypoglycemic peptide according to claim 1, characterized in that: The freezing and thawing of pig blood was carried out by adding anticoagulant to fresh pig blood, freezing it at -40°C overnight, thawing it at room temperature, and repeating the freezing and thawing process three times.

3. The method for preparing a porcine blood hypoglycemic peptide according to claim 1, characterized in that: The denaturation treatment of pig blood is as follows: the pig blood obtained after freezing and thawing is mixed evenly with water in a mass ratio of 1:3, and then the mixture is placed in a water bath at 90-95° C. for 30-60 minutes to obtain a pig blood solution.

4. The method for preparing a porcine blood hypoglycemic peptide according to claim 1, characterized in that: The alkaline protease was subjected to enzymatic hydrolysis at a pH of 7.5-8.5 and a temperature of 50°C. After the enzymatic hydrolysis was completed, the product was boiled for 10 minutes to inactivate the enzyme, and the pH was adjusted to 7 after cooling.

5. The method for preparing a porcine blood hypoglycemic peptide according to claim 1, characterized in that: The complex protease includes endoproteinase, exoproteinase and flavor enzyme.

6. The method for preparing a porcine blood hypoglycemic peptide according to claim 1 or claim 5, characterized in that: The composite protease was subjected to enzymatic hydrolysis at 50°C for 1-4 hours. After the enzymatic hydrolysis was completed, the mixture was boiled for 10 minutes to inactivate the enzyme, and the pH value was adjusted to 4.5 after cooling.

7. The method for preparing a porcine blood hypoglycemic peptide according to claim 1, characterized in that: The purification process is as follows: after centrifuging the composite enzymatic hydrolysate A, the supernatant is taken, the pH value is adjusted to 7.0, and then filtered using a 0.25 μm filter membrane. The filtrate is ultrafiltered in sequence using 10 KDa, 5 KDa and 3 KDa ultrafiltration membranes at 4°C to obtain a composite enzymatic hydrolysate B. The composite enzymatic hydrolysate B is vacuum freeze-dried to obtain the porcine blood hypoglycemic peptide.

8. The method for preparing a porcine blood hypoglycemic peptide according to claim 1 or claim 7, characterized in that: The centrifugal speed is 6000-10000r / min, and the centrifugal time is 30-60min.

9. A porcine blood hypoglycemic peptide, characterized in that: Prepared by the preparation method as claimed in claim 1.

10. Use of the pig blood hypoglycemic peptide as claimed in claim 9 in the preparation of medicines or health foods that help maintain healthy blood sugar levels.

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