CN106676153A - Preparation method and application of meretrix lusoria blood-pressure-reducing peptide - Google Patents

Abstract

The invention provides a preparation method and application of a clam hypotensive peptide, the steps are as follows: pretreatment: shell the clam and take the meat and soak it in deionized water, remove and drain, homogenate, add distilled water and hydrochloric acid solution to adjust the pH; Enzymolysis: add 1.1-1.5% by weight of clam meat for enzymolysis with neutral protease, add 0.15-0.3% by weight of clam meat to obtain clam meat enzymatic hydrolysis solution; gel chromatography: enzymatically hydrolyze clam meat The solution was subjected to enzyme inactivation, centrifuged, and the supernatant was taken, and 1-3% by weight of clam meat was added to the eluent, left to stand, ultrafiltration, and gel chromatography to obtain clam hypotensive peptide concentrate; freeze-dried and stored. The invention has simple preparation process, short pretreatment time, high blood pressure-lowering peptide content in the enzymatic hydrolysis solution, high blood-pressure-lowering peptide content and extraction rate, and the extracted blood-pressure-lowering peptide can be used in medicine and food with blood pressure-lowering effect.

Description

Preparation method and application of clam hypotensive peptide

technical field

The invention belongs to the field of biotechnology and functional health care, and in particular relates to a preparation method and application of a clam hypotensive peptide.

Background technique

Hypertension is a common cardiovascular disease. It not only has a high prevalence rate, but also often causes serious heart, brain, kidney and other diseases. It is a major risk factor for stroke, coronary heart disease, heart failure and kidney disease. Although the current chemically synthesized antihypertensive drugs, such as Lisinopril and Perindopril, have obvious effects in treating hypertension, long-term use can easily cause adverse reactions such as skin rash and proteinuria.

Antihypertensive peptides, also known as angiotensin converting enzyme (Angiotensin I Converting Enzyme, ACE) inhibitory peptides, are a class of polypeptide short-chain substances isolated from food proteins that can significantly lower blood pressure. From the source, it can be divided into milk protein (including casein, whey protein), fermented food, animal protein, vegetable protein and natural ACE inhibitory peptide. These antihypertensive peptides derived from food are usually obtained by protease hydrolyzing protein under mild conditions, and are safe to eat. Their common outstanding advantage is that they only have a hypotensive effect on hypertensive patients, and have no antihypertensive effect on normal blood pressure. Therefore, there will be no excessive pressure reduction. In addition to the function of lowering blood pressure, they often have functions such as immune promotion, anticoagulation, easy digestion and absorption, and antitumor.

The existing technology provides a variety of methods and applications for extracting blood pressure-lowering peptides from organisms. For example, the Chinese invention patent document A sardine blood-lowering peptide and its preparation method and application authorization announcement number CN 104031967 B This invention belongs to food biotechnology and functions In the field of health care, a sardine hypotensive peptide and its preparation method and application are disclosed. The main technical steps of the method are as follows: after the sardines are pretreated into a homogenate, first add papain for enzymolysis, then add alkaline protease for enzymolysis; Centrifuging; taking the centrifuged supernatant, using ultrafiltration and gel chromatography to separate and purify the crude product; freeze-drying the separated and purified high-efficiency components to obtain the sardine hypotensive peptide. The yield of sardines is high and the price is low. Therefore, using sardines as raw materials to prepare hypotensive peptides can fully improve the comprehensive utilization value of sardines, and the sardine hypotensive peptides obtained by enzymatic hydrolysis have no safety problems and no chemical antihypertensive drugs. Side effects, antihypertensive effect ideal.

In the prior art, sardine hypotensive peptides are obtained by enzymatic hydrolysis, which has no side effects of chemical antihypertensive drugs and improves the comprehensive utilization value of sardines. However, the pretreatment time is too long, and two-step enzymatic hydrolysis is performed, and no improvement is made after obtaining the supernatant. The steps of precipitation of hypotensive peptides, enzymatic hydrolysis and extraction effects need to be improved, and the content of hypotensive peptides obtained needs to be improved.

Contents of the invention

Aiming at the above technical problems, the present invention provides a preparation method and application of a clam hypotensive peptide with short pretreatment time, high content of hypotensive peptide in enzymatic hydrolysis solution, and improved extraction rate of hypotensive peptide.

The solution adopted by the present invention for the above-mentioned technical problems is: a preparation method of clam hypotensive peptide, the preparation steps are as follows:

1) Pretreatment: remove the clams and take the meat and soak them in deionized water for 8-12 hours, remove and drain, homogenate, add distilled water 3-6 times the volume of clam meat and hydrochloric acid solution to adjust the pH to 6.5-8.5; Soak in deionized water to remove impurities, bacteria and salt in the clam. The deionized water used can shorten the soaking time and thus the extraction time, and achieve a good soaking effect. The added distilled water and hydrochloric acid solution quickly adjust the pH of the clam meat to The specified range is prepared for enzymatic hydrolysis, and the pretreatment time is obviously shortened by the prepared deionized water and hydrochloric acid solution, and the pretreatment effect is excellent.

2) Enzymolysis: adding 1.1-1.5% by weight of clam meat with neutral protease at 40-50°C for 1-2 hours, adding 0.15-0.3% by weight of clam meat with compound enzyme at 40-50°C Enzymatic hydrolysis for 1~3 hours, the enzymatic hydrolyzate of clam meat is obtained; the conventional one-time enzymatic hydrolysis is limited by the specific effect of the added enzyme, and the yield of blood pressure-lowering peptide is not ideal, and the method of two enzymatic hydrolysis and compound enzyme can significantly Improving the yield of hypotensive peptides can also ensure that other nutrients in clam meat are not lost, the enzymatic hydrolysis time is shortened but the effect is significantly improved, and the amount of enzymes is also saved, which can effectively save costs.

3) Gel chromatography: deactivate the clam meat enzymatic hydrolyzate at a temperature of 90-95°C for 5-10 minutes, and centrifuge with a centrifuge at a speed of 3000-5000r/min. 5 ~ 15min, get the supernatant, add clam meat 1 ~ 3% by weight precipitation agent, leave standstill, adopt ultrafiltration membrane to separate and purify the supernatant after adding the precipitation agent, the ultrafiltration membrane is that the molecular weight cut off is 3000~10000Da ultrafiltration membrane, gel chromatography, to obtain clam hypotensive peptide concentrate; inactivate the enzyme to make protein and hypotensive peptide aggregate and precipitate, prepare for centrifugation, add precipitation agent to the supernatant obtained after centrifugation , there may be some antihypertensive peptides in the supernatant that have not been extracted. Use a precipitant to fully extract this part of antihypertensive peptides to increase the extraction rate of antihypertensive peptides, and then use ultrafiltration and gel chromatography to obtain high The pure antihypertensive peptide does not need to add any chemical reagents and will not cause changes in temperature and pH, so it will not affect the purity of the antihypertensive peptide, and can prevent the inactivation and autolysis of the antihypertensive peptide.

4) Freeze-dried and stored. The freeze-drying method used is a drying process that dehydrates the blood pressure-lowering peptide at low temperature and low pressure. Compared with other drying methods, it has the advantages of no deterioration of the blood-pressure-lowering peptide, easy long-term storage, complete dehydration, and less loss of volatile components.

The compound enzyme in step 2 is composed of the following components and parts by weight: 10-15 parts of papain, 3-8 parts of cellulase, 1-4 parts of pectinase, 1-2 parts of α1-antichymotrypsin and adenylate ring Lyase 0.3~1 part and γ-glutamyl transferase 0.5~0.8 part. It can significantly increase the yield of blood pressure-lowering peptides, and can also ensure that other nutrients in clam meat are not lost, the enzymatic hydrolysis time is shortened but the effect is significantly improved, and the amount of enzymes is also saved, which can effectively save costs.

The deionized water in step 1 contains 0.9-1.1 parts of glacial acetic acid, 0.9-1.1 parts of ethanol, 1.8-2.2 parts of sulfated oil, 1-2 parts of 4-fluoroacetanilide and 0.3-0.7 parts of n-propyl nitrate. The impurities, bacteria and salt in clams are removed by immersion in deionized water. The deionized water used can shorten the immersion time and thus the extraction time, and achieve a good immersion effect.

In step 2, the hydrochloric acid solution is added with 2-5 parts by weight of sodium hydroxide and 1-3 parts by weight of 3-ethoxy-4-hydroxybenzaldehyde. Quickly adjust the pH of clam meat to the specified range to prepare for enzymatic hydrolysis and save extraction time.

In step 3, the precipitation agent is composed of the following components and parts by weight: 3 to 5 parts of ammonium sulfate, 1 to 3 parts of sodium chloride, 0.5 to 1 part of potassium nitrate, 0.2 to 0.4 parts of pentaerythritol, and 0.05 to 0.1 parts of sodium dichloroisocyanurate parts and 0.7~0.8 parts of magnesium hypophosphite. There may be some antihypertensive peptides in the supernatant that have not been extracted, and the eluting agent is used to fully extract this part of the antihypertensive peptides, and the above composition has a certain activity retention effect, which indirectly increases the extraction rate of the antihypertensive peptides .

The clam blood pressure lowering peptide obtained by extracting the blood pressure lowering peptide from clams is used in the preparation of medicines and foods with blood pressure lowering effects.

Compared with the prior art, the advantage of this method is that the deionized water used can shorten the immersion time and thus the extraction time, and achieve a good immersion effect, and the added distilled water cooperates with the hydrochloric acid solution to quickly adjust the pH of clam meat to a specified range , the pretreatment time is obviously shortened by the prepared deionized water and hydrochloric acid solution, and the pretreatment effect is excellent. Using two enzymatic hydrolysis and compound enzyme method can significantly increase the yield of hypotensive peptides, and can also ensure that other nutrients in Meretrix meretrix are not lost, the enzymatic hydrolysis time is shortened but the effect is significantly improved, and the amount of enzymes is also saved. Can effectively save costs. Add an eluent to the supernatant obtained after centrifugation, and there may be some blood pressure-lowering peptides in the supernatant that have not been extracted. Use the eluent to fully extract this part of the blood-pressure-lowering peptide to increase the extraction rate of the blood-pressure-lowering peptide. Using ultrafiltration and gel chromatography to obtain high-purity blood pressure-lowering peptides, and does not need to add any chemical reagents and will not cause changes in temperature and pH, so it will not affect the purity of blood-pressure-lowering peptides and prevent blood-pressure-lowering peptides from losing Living, autolysis and other issues. The preparation process of the present invention is simple, the pretreatment time is short, the content of hypotensive peptide in the enzymatic hydrolysis solution is high, the content and extraction rate of hypotensive peptide are improved, and the extracted hypotensive peptide can be used in medicines and foods with hypotensive effect, which can reduce blood pressure Peptides are safe to eat, and can lower blood pressure for hypertensive patients, but have no antihypertensive effect for normal blood pressure patients, and there will be no transitional phenomenon of lowering blood pressure.

specific embodiment

Describe in further detail below in conjunction with specific embodiment:

Example 1:

A preparation method of clam hypotensive peptide, the preparation steps are as follows:

1) Pretreatment: remove the clams and take the meat and soak them in deionized water for 8-12 hours, remove and drain, homogenate, add distilled water 3-6 times the volume of clam meat and hydrochloric acid solution to adjust the pH to 6.5-8.5; Soak in deionized water to remove impurities, bacteria and salt in the clam. The deionized water used can shorten the soaking time and thus the extraction time, and achieve a good soaking effect. The added distilled water and hydrochloric acid solution quickly adjust the pH of the clam meat to The specified range is prepared for enzymatic hydrolysis, and the pretreatment time is obviously shortened by the prepared deionized water and hydrochloric acid solution, and the pretreatment effect is excellent.

2) Enzymolysis: adding 1.1-1.5% by weight of clam meat with neutral protease at 40-50°C for 1-2 hours, adding 0.15-0.3% by weight of clam meat with compound enzyme at 40-50°C Enzymatic hydrolysis for 1~3 hours, the enzymatic hydrolyzate of clam meat is obtained; the conventional one-time enzymatic hydrolysis is limited by the specific effect of the added enzyme, and the yield of blood pressure-lowering peptide is not ideal, and the method of two enzymatic hydrolysis and compound enzyme can significantly Improving the yield of hypotensive peptides can also ensure that other nutrients in clam meat are not lost, the enzymatic hydrolysis time is shortened but the effect is significantly improved, and the amount of enzymes is also saved, which can effectively save costs.

3) Gel chromatography: deactivate the clam meat enzymatic hydrolyzate at a temperature of 90-95°C for 5-10 minutes, and centrifuge with a centrifuge at a speed of 3000-5000r/min. 5 ~ 15min, get the supernatant, add clam meat 1 ~ 3% by weight precipitation agent, leave standstill, adopt ultrafiltration membrane to separate and purify the supernatant after adding the precipitation agent, the ultrafiltration membrane is that the molecular weight cut off is 3000~10000Da ultrafiltration membrane, gel chromatography, to obtain clam hypotensive peptide concentrate; inactivate the enzyme to make protein and hypotensive peptide aggregate and precipitate, prepare for centrifugation, add precipitation agent to the supernatant obtained after centrifugation , there may be some antihypertensive peptides in the supernatant that have not been extracted. Use a precipitant to fully extract this part of antihypertensive peptides to increase the extraction rate of antihypertensive peptides, and then use ultrafiltration and gel chromatography to obtain high The pure antihypertensive peptide does not need to add any chemical reagents and will not cause changes in temperature and pH, so it will not affect the purity of the antihypertensive peptide, and can prevent the inactivation and autolysis of the antihypertensive peptide.

4) Freeze-dried and stored. The freeze-drying method used is a drying process that dehydrates the blood pressure-lowering peptide at low temperature and low pressure. Compared with other drying methods, it has the advantages of no deterioration of the blood-pressure-lowering peptide, easy long-term storage, complete dehydration, and less loss of volatile components.

The compound enzyme in step 2 is composed of the following components and parts by weight: 10-15 parts of papain, 3-8 parts of cellulase, 1-4 parts of pectinase, 1-2 parts of α1-antichymotrypsin and adenylate ring Lyase 0.3~1 part and γ-glutamyl transferase 0.5~0.8 part. It can significantly increase the yield of blood pressure-lowering peptides, and can also ensure that other nutrients in clam meat are not lost, the enzymatic hydrolysis time is shortened but the effect is significantly improved, and the amount of enzymes is also saved, which can effectively save costs.

The deionized water in step 1 contains 0.9-1.1 parts of glacial acetic acid, 0.9-1.1 parts of ethanol, 1.8-2.2 parts of sulfated oil, 1-2 parts of 4-fluoroacetanilide and 0.3-0.7 parts of n-propyl nitrate. The impurities, bacteria and salt in clams are removed by immersion in deionized water. The deionized water used can shorten the immersion time and thus the extraction time, and achieve a good immersion effect.

In step 2, the hydrochloric acid solution is added with 2-5 parts by weight of sodium hydroxide and 1-3 parts by weight of 3-ethoxy-4-hydroxybenzaldehyde. Quickly adjust the pH of clam meat to the specified range to prepare for enzymatic hydrolysis and save extraction time.

In step 3, the precipitation agent is composed of the following components and parts by weight: 3 to 5 parts of ammonium sulfate, 1 to 3 parts of sodium chloride, 0.5 to 1 part of potassium nitrate, 0.2 to 0.4 parts of pentaerythritol, and 0.05 to 0.1 parts of sodium dichloroisocyanurate parts and 0.7~0.8 parts of magnesium hypophosphite. There may be some antihypertensive peptides in the supernatant that have not been extracted, and the eluting agent is used to fully extract this part of the antihypertensive peptides to increase the extraction rate of the antihypertensive peptides.

The clam blood pressure lowering peptide obtained by extracting the blood pressure lowering peptide from clams is used in the preparation of medicines and foods with blood pressure lowering effects.

Example 2:

A method for preparing clam hypotensive peptide, the preferred preparation steps are as follows:

1) Pretreatment: Remove the shells and take the meat of clams and soak them in deionized water for 12 hours, remove and drain, homogenate, add distilled water and hydrochloric acid solution preferably 5 times the volume of clams meat to adjust the pH to 7; Soak in deionized water to remove impurities, bacteria and salt in the clam. The deionized water used can shorten the soaking time and thus the extraction time, and achieve a good soaking effect. The added distilled water and hydrochloric acid solution quickly adjust the pH of the clam meat to The specified range is prepared for enzymatic hydrolysis, and the pretreatment time is obviously shortened by the prepared deionized water and hydrochloric acid solution, and the pretreatment effect is excellent.

2) Enzymolysis: adding preferred neutral protease at 1.1% by weight of preferred clam meat for optimal enzymolysis at 45°C for 2 hours, adding preferred compound enzyme with preferred clam meat at 0.18% by weight for optimal enzymolysis at 45°C for 1 hour, Obtain clam meat enzymatic hydrolysis solution; conventional one-time enzymatic hydrolysis is limited by the specific effect of adding enzymes, and the yield of hypotensive peptides is not ideal, and the method of two enzymatic hydrolysis and compound enzymes can significantly increase the yield of hypotensive peptides The efficiency can also ensure that other nutrients in the clam meat are not lost, the enzymatic hydrolysis time is shortened but the effect is significantly improved, and the amount of enzymes is also saved, which can effectively save costs.

3) Gel chromatography: deactivate the clam meat enzymatic hydrolyzate, the enzyme inactivation temperature is preferably 92°C, the time is preferably 6min, and centrifuged with a centrifuge, preferably the centrifugal speed is 4000r/min, and the centrifugation time is preferably 10min, Get the supernatant, add preferred clam meat 2% weight part precipitation agent, leave standstill, adopt the ultrafiltration membrane to separate and purify the supernatant after adding the precipitation agent, and the ultrafiltration membrane is preferably an ultrafiltration with a molecular weight cut-off of 7000Da Membrane, gel chromatography, to obtain clam hypotensive peptide concentrate; inactivate the enzyme to make protein and hypotensive peptide aggregate and precipitate, prepare for centrifugation, add eluent to the supernatant obtained after centrifugation, and add eluent to the supernatant There may be some antihypertensive peptides that have not been extracted. Use the eluent to fully extract this part of the antihypertensive peptides to increase the extraction rate of the antihypertensive peptides, and then use ultrafiltration and gel chromatography to obtain high-purity antihypertensive peptides. Moreover, it does not need to add any chemical reagents and will not cause changes in temperature and pH, so the purity of the hypotensive peptide will not be affected, and problems such as inactivation and autolysis of the hypotensive peptide can be prevented.

4) Freeze-dried and stored. The freeze-drying method used is a drying process that dehydrates the blood pressure-lowering peptide at low temperature and low pressure. Compared with other drying methods, it has the advantages of no deterioration of the blood-pressure-lowering peptide, easy long-term storage, complete dehydration, and less loss of volatile components.

The compound enzyme in step 2 is composed of the following components and preferred parts by weight: preferably 13 parts of papain, preferably 7 parts of cellulase, preferably 3 parts of pectinase, preferably 1 part of α1-antichymotrypsin and preferably adenylate cyclase 0.5 parts and preferably 0.6 parts of gamma-glutamyltransferase. It can significantly increase the yield of blood pressure-lowering peptides, and can also ensure that other nutrients in clam meat are not lost, the enzymatic hydrolysis time is shortened but the effect is significantly improved, and the amount of enzymes is also saved, which can effectively save costs.

The deionized water in step 1 contains preferably 1 part of glacial acetic acid, preferably 1 part of ethanol, preferably 1.9 parts of sulfated oil, preferably 1 part of 4-fluoroacetanilide and preferably 0.5 part of n-propyl nitrate. The impurities, bacteria and salt in clams are removed by immersion in deionized water. The deionized water used can shorten the immersion time and thus the extraction time, and achieve a good immersion effect.

In step 2, the hydrochloric acid solution is preferably added with 3 parts by weight of sodium hydroxide and 2 parts by weight of 3-ethoxy-4-hydroxybenzaldehyde. Quickly adjust the pH of clam meat to the specified range to prepare for enzymatic hydrolysis and save extraction time.

In step 3, the eluting agent is composed of the following components and preferred parts by weight: preferably 4 parts of ammonium sulfate, preferably 2 parts of sodium chloride, preferably 0.7 part of potassium nitrate, preferably 0.3 part of pentaerythritol, preferably 0.07 part of sodium dichloroisocyanurate and preferably 0.75 parts of magnesium phosphate. There may be some antihypertensive peptides in the supernatant that have not been extracted, and the eluting agent is used to fully extract this part of the antihypertensive peptides to increase the extraction rate of the antihypertensive peptides.

The clam blood pressure lowering peptide obtained by extracting the blood pressure lowering peptide from clams is used in the preparation of medicines and foods with blood pressure lowering effects.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplification should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (9)

1. A preparation method of clam hypotensive peptide, characterized in that the following steps: 1) Pretreatment: remove the shells of Meretrix meretrix and soak the meat in deionized water, remove and drain, homogenate, add distilled water and hydrochloric acid solution to adjust the pH; 2) Enzymolysis: add 1.1-1.5% by weight of clam meat for enzymolysis with neutral protease, and add 0.15-0.3% by weight of clam meat for enzymatic hydrolysis to obtain clam meat enzymatic hydrolysis solution; 3) Gel chromatography: deactivate the enzyme hydrolyzate of meretrix meretrix, centrifuge it, take the supernatant, add 1-3% by weight of clam meat eluent, let stand, perform ultrafiltration, gel chromatography, and obtain clam meretrix Antihypertensive peptide concentrate; 4) Freeze-dried and stored. 2. The preparation method of a clam hypotensive peptide according to claim 1, characterized in that: in step 1, Chinese clam meat is soaked in deionized water for 8-12 hours, and distilled water is added to 3-6 times the volume of clam meat, Add hydrochloric acid solution to adjust the pH to 6.5~8.5. 3. The preparation method of clam hypotensive peptide according to claim 1, characterized in that: in the step 2, neutral protease is added for enzymolysis at 40~50°C for 1~2h, and compound enzyme is added at 40 Enzymatic hydrolysis at ~50°C for 1~3h. 4. The preparation method of a clam hypotensive peptide according to claim 1, characterized in that: the compound enzyme in said step 2 is composed of the following components and parts by weight: 10~15 parts of papain, 3~15 parts of cellulase 8 parts, 1~4 parts of pectinase, 1~2 parts of α1-antichymotrypsin, 0.3~1 part of adenylate cyclase and 0.5~0.8 parts of γ-glutamyltransferase. 5. The preparation method of a clam hypotensive peptide according to claim 1, characterized in that: in the step 2, the hydrochloric acid solution is added with 2 to 5 parts by weight of sodium hydroxide and 3-ethoxy-4-hydroxyl Benzaldehyde 1 ~ 3 parts by weight. 6. The preparation method of clam hypotensive peptide according to claim 1, characterized in that: the deionized water in the step 1 contains 0.9~1.1 parts of glacial acetic acid, 0.9~1.1 parts of ethanol, and 1.8~1 parts of sulfated oil. 2.2 parts, 1~2 parts of 4-fluoroacetanilide and 0.3~0.7 parts of n-propyl nitrate. 7. The preparation method of a clam hypotensive peptide according to claim 1, characterized in that: in the step 3, the enzyme inactivation temperature is 90~95°C, and the time is 5~10min; The centrifuge is centrifuged, the centrifugal speed is 3000 ~ 5000r/min, and the centrifugation time is 5 ~ 15min; in the step 3, an ultrafiltration membrane is used to separate and purify the supernatant after adding the precipitation agent, and the ultrafiltration membrane is a molecular weight cut-off of 3000~10000Da ultrafiltration membrane. 8. The preparation method of clam hypotensive peptide according to claim 1, characterized in that: the eluting agent in the step 3 is composed of the following components and parts by weight: 3~5 parts of ammonium sulfate, 1~5 parts of sodium chloride 3 parts, 0.5~1 part of potassium nitrate, 0.2~0.4 part of pentaerythritol, 0.05~0.1 part of sodium dichloroisocyanurate and 0.7~0.8 part of magnesium hypophosphite. 9. The application of a clam hypotensive peptide according to claim 1 for preparing health food or food additive with hypotensive effect.