Protein nucleus yellow-promoting algae antihypertensive peptide as well as preparation method and application thereof

Abstract

The invention discloses a xanthoceralcaryon antihypertensive peptide as well as a preparation method and application thereof, the amino acid sequences of the antihypertensive peptide are Asp-Gly-Arg-Pro-Gly-Pro, Asp-Gly-Leu-Asp-Gly-Leu-Arg and Gly-Pro-Pro-Ala-Gly-Pro-Glu-Gly-Arg-Pro respectively, and the corresponding ESI / MS detection molecular weights of the antihypertensive peptide are 597.287 Da, 744.3766 Da and 836.414 Da respectively. The xanthomonas pyrenotrophora antihypertensive peptide prepared by the invention has obvious angiotensin converting enzyme (ACE) inhibitory activity, and can be applied to drugs or health food related to hypertension treatment. The antihypertensive peptide with an ACE (angiotensin converting enzyme) inhibition effect is prepared by taking the crude protein powder of the xanthoceras pyrenotrophora as a raw material and utilizing an enzymolysis process and an ultrafiltration technology, and technical support is provided for high-value utilization of the xanthoceras pyrenotrophora.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptides, and in particular to a protein core-stimulating yellow algae blood pressure lowering peptide and a preparation method and application thereof. Background Art

[0002] Hypertension is a common and frequently occurring disease. The disease generally develops slowly, and patients are often asymptomatic in the early stage, or only have symptoms such as dizziness, headache, palpitations, and tinnitus. On the surface, it is an independent disease, but in fact it is an important risk factor for cardiovascular, cerebrovascular and renal diseases. If improperly treated, it will become a more serious cerebral stroke, myocardial infarction and renal failure and other common complications of hypertension. Therefore, the development of effective drugs and health foods for the treatment of hypertension has great prospects. In the process of blood pressure regulation, angiotensin converting enzyme (ACE) can catalyze the degradation of angiotensin I into angiotensin II on the one hand, and on the other hand, it can catalyze the degradation of bradykinin. Angiotensin II binds to angiotensin II type I receptors to cause vasoconstriction and water-sodium reabsorption, thereby causing blood pressure to rise. At the same time, bradykinin degradation can also increase blood pressure. Therefore, ACE inhibitors have always been the first-line drugs for the treatment of hypertension. However, due to the side effects of existing ACE inhibitors, such as dry cough, rash, hypokalemia, etc., the development of more efficient and safe ACE inhibitor compounds is still a research hotspot. Summary of the invention

[0003] In view of the technical problem that existing ACE inhibitors have obvious side effects such as dry cough, rash, hypokalemia, etc., the present invention provides a pyrenoidosa antihypertensive peptide and a preparation method and application thereof. Using pyrenoidosa as raw material, an enzymatic hydrolysis process and ultrafiltration technology are used to prepare a antihypertensive peptide capable of inhibiting ACE, providing technical support for the high-value utilization of pyrenoidosa.

[0004] The amino acid sequences of the protein core-stimulating yellow algae blood pressure-lowering peptides provided by the present invention are: Asp-Gly-Arg-Pro-Gly-Pro (abbreviated as DGRPGP), Asp-Gly-Leu-Asp-Gly-Leu-Arg (abbreviated as DGLDGLR) and Gly-Pro-Ala-Gly-Pro-Glu-Gly-Arg-Pro (abbreviated as GPAGPEGRP), the corresponding ESI / MS detected molecular weights are 597.287 Da, 744.3766 Da and 836.414 Da, respectively.

[0005] The preparation method of the above-mentioned pyrenoid algae antihypertensive peptide comprises the steps of pretreatment of pyrenoid algae crude protein powder, enzymatic hydrolysis and ultrafiltration purification.

[0006] Furthermore, the preparation method of the protein core-stimulating yellow algae antihypertensive peptide comprises the following steps: S1. Pretreatment of crude protein powder of pyrenoid algae: dissolving the crude protein powder of pyrenoid algae in water, mixing, centrifuging to obtain the supernatant, adding ammonium sulfate solution to precipitate protein, continuing centrifugation, removing the supernatant, dissolving the precipitate in water, dialysis and freeze-drying; freeze-drying to obtain pyrenoid algae protein powder; S2. Enzymatic hydrolysis: Mix the protein powder of the pyrenoid algae obtained in S1 with water, adjust the temperature to 25-45°C, add flavor protease, and perform enzymatic hydrolysis for 2-6 hours, with a pH of 6-8; place the enzymatic hydrolyzate in boiling water for 8-15 minutes to inactivate the enzyme, and obtain the pyrenoid algae enzymatic hydrolyzate; S3. Ultrafiltration purification: The enzymatic hydrolysate is ultrafiltered and centrifuged to collect samples with molecular weight less than 3kDa and greater than or equal to 3kDa, respectively, and the angiotensin converting enzyme (ACE) inhibitory activity of each component is determined. The optimal ultrafiltration component is selected to obtain the protein core chlorophyll antihypertensive peptide.

[0007] Furthermore, in S1, distilled water is used, and the weight-to-volume ratio of the crude protein powder of pyrenoidosa to distilled water is 1:20-30 g / mL.

[0008] Further, in S1, the Latin name of Pyrenophyte is Chlorella pyrenoidosa.

[0009] Furthermore, in S1, the mass fraction of the ammonium sulfate solution is 70% to 80%.

[0010] Furthermore, in S1, the dialysis time was 36–48 h.

[0011] Furthermore, in S1, distilled water is used, and the weight-to-volume ratio of the pyrenoidosa protein powder to the distilled water is 1:80-100 g / mL.

[0012] Furthermore, in S2, the amount of flavor protease used is 13% to 21% of the mass of the pyrenoid chlorophyll protein powder.

[0013] Furthermore, in S3, the ultrafiltration speed is 5000-6000 rpm, and the time is 10-20 min.

[0014] The above-mentioned protein core-promoting yellow algae antihypertensive peptide is used for the preparation of antihypertensive drugs or health foods, and can exert good angiotensin converting enzyme (ACE) inhibitory activity.

[0015] The beneficial effects of the present invention are: (1) The present invention uses crude protein powder of pyrenoid algae as raw material, and uses enzymatic hydrolysis and ultrafiltration technology to prepare antihypertensive peptides with ACE inhibitory effect, providing technical support for the high-value utilization of pyrenoid algae. The preparation method adopted by the present invention is a controllable and environmentally friendly bioenzymatic method. It is easy to release the pyrenoid algae antihypertensive peptide to the greatest extent by monitoring the enzymatic hydrolysis process, thereby improving the utilization rate of the raw materials; at the same time, the prepared antihypertensive peptide is safe and has no toxic side effects.

[0016] (2) The antihypertensive peptide prepared by the present invention has significant angiotensin converting enzyme (ACE) inhibitory activity and can be used in the preparation of drugs or health foods related to the treatment of hypertension. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the inhibition rate of angiotensin converting enzyme before and after protein hydrolysis of the protease of the pyrenoidosa and before and after ultrafiltration of the hydrolyzate at a concentration of 3 mg / mL in Example 1.

[0018] Figure 2 This is the structural diagram of the protein core chlorophyll antihypertensive peptide Asp-Gly-Arg-Pro-Gly-Pro obtained in Example 1.

[0019] Figure 3 This is the mass spectrum of the protein core chlorophyll antihypertensive peptide Asp-Gly-Arg-Pro-Gly-Pro obtained in Example 1.

[0020] Figure 4 This is the structural diagram of the protein core chlorophyll antihypertensive peptide Asp-Gly-Leu-Asp-Gly-Leu-Arg obtained in Example 1.

[0021] Figure 5 This is the mass spectrum of the protein core chlorophyll antihypertensive peptide Asp-Gly-Leu-Asp-Gly-Leu-Arg obtained in Example 1.

[0022] Figure 6 This is the structural diagram of the protein core chlorophyll antihypertensive peptide Gly-Pro-Ala-Gly-Pro-Glu-Gly-Arg-Pro obtained in Example 1.

[0023] Figure 7 This is the mass spectrum of the protein core chlorophyll antihypertensive peptide Gly-Pro-Ala-Gly-Pro-Glu-Gly-Arg-Pro obtained in Example 1.

[0024] Figure 8 , 9This is the IC50 chart of the protein core chlorophyll antihypertensive peptides Asp-Gly-Arg-Pro-Gly-Pro, Asp-Gly-Leu-Asp-Gly-Leu-Arg and Gly-Pro-Ala-Gly-Pro-Glu-Gly-Arg-Pro obtained in Example 1. DETAILED DESCRIPTION

[0025] The following examples are used to further illustrate the present invention, but they do not limit or define the scope of the present invention. The raw material used in the experiment of the present invention is crude protein powder of Pyrenophyte. Example 1

[0026] (1) Pretreatment of crude protein powder of Pyrenophyte: Mix crude protein powder of Pyrenophyte with distilled water in a mass volume ratio of 1 g:20 mL, stir evenly, centrifuge to obtain supernatant, add 80% ammonium sulfate solution by mass to precipitate protein, continue centrifugation to obtain precipitate, add water to dissolve, put into dialysis bag for 48 hours, change water every four hours, and freeze-dry to obtain Pyrenophyte protein powder.

[0027] (2) Enzymatic hydrolysis: the above-mentioned pyrenoid algae protein powder was mixed with distilled water at a mass volume ratio of 1 g:100 mL, the temperature was adjusted to 43.4° C., flavor protease was added at 21% of the mass of the pyrenoid algae protein powder, and enzymatic hydrolysis was performed at pH 7.2 for 5.8 h; the enzymatic hydrolyzate was placed in boiling water for 10 min to inactivate the enzyme, and the pyrenoid algae protein hydrolyzate was obtained; (3) Ultrafiltration purification: The protein core-stimulating algae protein hydrolysate was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and the components with molecular weights less than 3 kDa and greater than or equal to 3 kDa were collected respectively. The inhibition rate of each component on angiotensin converting enzyme at a concentration of 3 mg / mL was determined. The results were as follows: Figure 1 As shown, the results showed that the fraction with a molecular weight less than 3 kDa had the highest ACE inhibitory activity, so the fraction with a molecular weight less than 3 kDa was used for sequence identification; Sequence identification: The components with the highest ACE inhibition rate less than 3 kDa were collected and the amino acid sequences were determined by protein / peptide sequence analyzer to be Asp-Gly-Arg-Pro-Gly-Pro (DGRPGP), Asp-Gly-Leu-Asp-Gly-Leu-Arg (DGLDGLR) and Gly-Pro-Ala-Gly-Pro-Glu-Gly-Arg-Pro (GPAGPEGRP). The molecular structure and mass spectrum are shown in the figure. Figures 2 to 7As shown. ESI / MS detected the molecular weights of the above three peptides to be 597.287Da, 744.3766Da and 836.414Da, respectively. The IC50 values ​​of the three peptides were determined to be 0.00289mg / mL, 0.3668 mg / mL and 0.00118 mg / mL, respectively (as shown Figure 8 and Fig. 9 as shown). Example 2

[0028] (1) Pretreatment of crude protein powder of Pyrenophyte: Mix crude protein powder of Pyrenophyte with distilled water in a weight-to-volume ratio of 1 g:30 mL, stir evenly, centrifuge to obtain supernatant, add 75% by mass ammonium sulfate solution to precipitate protein, continue centrifugation to obtain precipitate, add water to dissolve, put into dialysis bag for 48 h, change water every four hours, and freeze-dry to obtain Pyrenophyte protein powder.

[0029] (2) Enzymatic hydrolysis: The above-mentioned pyrenoid algae protein powder was mixed with distilled water in a weight-volume ratio of 1 g:90 mL, the temperature was adjusted to 45° C., and flavor protease was added in an amount of 13% by weight of the pyrenoid algae protein powder. The enzymatic hydrolysis was performed at pH 7.5 for 6 h; the enzymatic hydrolyzate was placed in boiling water for 15 min to inactivate the enzyme, thereby obtaining a pyrenoid algae protein hydrolyzate; (3) Ultrafiltration purification: The protein core-stimulating algae protein hydrolysate was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and the components with molecular weights less than 3 kDa and greater than or equal to 3 kDa were collected respectively. The angiotensin converting enzyme inhibition rate of each component at a concentration of 3 mg / mL was determined. The component less than 3 kDa had the highest ACE inhibitory activity and was used for sequence identification; Sequence identification: The components with the highest ACE inhibition rate were collected and the amino acid sequences were determined by protein / peptide sequence analyzer to be Asp-Gly-Arg-Pro-Gly-Pro (DGRPGP), Asp-Gly-Leu-Asp-Gly-Leu-Arg (DGLDGLR) and Gly-Pro-Ala-Gly-Pro-Glu-Gly-Arg-Pro (GPAGPEGRP). The molecular weights detected by ESI / MS were 597.287Da, 744.3766Da and 836.414Da, respectively. Example 3

[0030] (1) Pretreatment of crude protein powder of Pyrenoidosa Chrysanthemi: Mix crude protein powder of Pyrenoidosa Chrysanthemi with distilled water in a weight-to-volume ratio of 1 g:25 mL, stir evenly, centrifuge to obtain supernatant, add 70% ammonium sulfate solution by mass to precipitate protein, continue centrifugation to obtain precipitate, add water to dissolve, put into dialysis bag for 36 h, change water every four hours, and freeze-dry to obtain Pyrenoidosa Chrysanthemi protein powder.

[0031] (2) Enzymatic hydrolysis: the above-mentioned pyrenoid algae protein powder was mixed with distilled water in a weight-volume ratio of 1 g:80 mL, the temperature was adjusted to 38° C., flavor protease was added in an amount of 18% by weight of the pyrenoid algae protein powder, and enzymatic hydrolysis was performed at pH 6.5 for 5 h; the enzymatic hydrolyzate was placed in boiling water for 8 min to inactivate the enzyme, and the pyrenoid algae protein hydrolyzate was obtained; (3) Ultrafiltration purification: The protein core-stimulating algae protein hydrolysate was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and the components with molecular weights less than 3 kDa and greater than or equal to 3 kDa were collected respectively. The angiotensin converting enzyme inhibition rate of each component at a concentration of 3 mg / mL was determined. The component less than 3 kDa had the highest ACE inhibitory activity and was used for sequence identification; Sequence identification: The components with the highest ACE inhibition rate were collected and the amino acid sequences were determined by protein / peptide sequence analyzer to be Asp-Gly-Arg-Pro-Gly-Pro (DGRPGP), Asp-Gly-Leu-Asp-Gly-Leu-Arg (DGLDGLR) and Gly-Pro-Ala-Gly-Pro-Glu-Gly-Arg-Pro (GPAGPEGRP). The molecular weights detected by ESI / MS were 597.287Da, 744.3766Da and 836.414Da, respectively.

Claims

1. A protein core-stimulating algae antihypertensive peptide, characterized in that: The amino acid sequences of the protein core-stimulating algae antihypertensive peptides are: Asp-Gly-Arg-Pro-Gly-Pro, Asp-Gly-Leu-Asp-Gly-Leu-Arg and Gly-Pro-Ala-Gly-Pro-Glu-Gly-Arg-Pro, the corresponding ESI / MS detected molecular weights were 597.287 Da, 744.3766 Da and 836.414 Da, respectively.

2. The method for preparing the pyrenoid chlorophyll hypotensive peptide according to claim 1, characterized in that: The method comprises pretreatment, enzymatic hydrolysis and ultrafiltration purification of crude protein powder of pyrenoidosa.

3. The method for preparing the pyrenoid chrysophageal antihypertensive peptide according to claim 2, characterized in that: The following steps are involved: S1. Pretreatment of crude protein powder of pyrenoid algae: dissolving the crude protein powder of pyrenoid algae in water, mixing, centrifuging to obtain the supernatant, adding ammonium sulfate solution to precipitate protein, continuing centrifugation, removing the supernatant, dissolving the precipitate in water, dialysis and freeze-drying; freeze-drying to obtain pyrenoid algae protein powder; S2. Enzymatic hydrolysis: Mix the protein powder of the pyrenoid algae obtained in S1 with water, adjust the temperature to 25-45°C, add flavor protease, and perform enzymatic hydrolysis for 2-6 hours, with a pH of 6-8; place the enzymatic hydrolyzate in boiling water for 8-15 minutes to inactivate the enzyme, and obtain the pyrenoid algae enzymatic hydrolyzate; S3. Ultrafiltration purification: The enzymatic hydrolysate is ultrafiltered and centrifuged to collect samples with molecular weight less than 3kDa and greater than 3kDa, respectively, and the angiotensin converting enzyme inhibitory activity of each component is determined. The ultrafiltration component with the best ACE inhibitory activity is selected to obtain the protein core chlorophyll antihypertensive peptide.

4. The method for preparing the pyrenoid chlorophyll hypotensive peptide according to claim 3, characterized in that: In S1, the water is distilled water, and the weight-to-volume ratio of the crude protein powder of pyrenoidosa to distilled water is 1:20-30 g / mL.

5. The method for preparing the pyrenoid chrysophageal antihypertensive peptide according to claim 3, characterized in that: In S1, the Latin name of the pyrenoidosa is Chlorella pyrenoidosa.

6. The method for preparing the pyrenoid chrysophageal antihypertensive peptide according to claim 3, characterized in that: In S1, the mass fraction of the ammonium sulfate solution is 70-80%; and the dialysis time is 36-48 h.

7. The method for preparing the pyrenoid chrysophageal antihypertensive peptide according to claim 3, characterized in that: In S2, the water is distilled water, and the weight-to-volume ratio of the pyrenoid chlorophyll protein powder to the distilled water is 1:80-100 g / mL.

8. The method for preparing the pyrenoid chrysophageal antihypertensive peptide according to claim 3, characterized in that: In S2, the amount of flavor protease used is 13%~21% of the mass of the pyrenoid chlorophyll protein powder.

9. The method for preparing the pyrenoid chrysophageal antihypertensive peptide according to claim 3, characterized in that: In S3, the ultrafiltration speed is 5000~6000rpm and the time is 10~20min.

10. Use of the pyrenoid chrysogenum antihypertensive peptide according to claim 1 or the pyrenoid chrysogenum antihypertensive peptide obtained by the preparation method according to any one of claims 2 to 9 in the preparation of antihypertensive drugs or health foods.

Images