WO2014194530A1 - Method for preparing ace inhibitory peptides from hairtail leftovers - Google Patents

Abstract

Disclosed in the present invention is a method for preparing ACE (Angiotensin-Converting Enzyme) inhibitory peptides from hairtail leftovers. The ACE inhibitory peptides are obtained by the steps of cleaning and mixing the hairtail leftovers with water, homogenization, enzymolysis and plate-and-frame filtration, intercepting small molecules of less than 3000 Da by ultrafiltration, and drying. Sephadex G-15 and RP-HPLC (Reverse Phase High-Performance Liquid Chromatography) can further be employed for two-step separation and purification to obtain high-purity ACE inhibitory peptides. The method of the present invention can change wastes into valuables by converting low-value hairtail leftovers into high-value ACE inhibitory peptides, achieve full use of resources, and has the advantages of easy process route, simplicity in operation, highly developed equipment used in the method, low investment cost and high recovery rate of the ACE inhibitory peptides.

Description

 Method for producing ACE inhibitory peptide by using fish waste

Technical field

The invention relates to the field of deep processing of food, in particular to deep processing of fish waste.

Background technique

Hypertension as a multi-cause, progressive cardiovascular disease, clinical manifestations of systemic active blood pressure, patients with systolic blood pressure ≥ 140 mmHg, diastolic blood pressure ≥90 mmHg. As the disease progresses, blood pressure continues to rise, followed by organ damage in the heart, brain, kidney and retina, leading to congestive heart failure, stroke, coronary heart disease, renal failure, aortic aneurysm morbidity and mortality. high. Hypertension is a disease with a high prevalence and has become a major public health problem worldwide. According to the press release issued by the International Society of Hypertension, people with high blood pressure or high blood pressure in the world have 972 million people, about the world’s adult population 26.4%. According to the World Health Organization (WHO) statistics on deaths of various diseases worldwide, the proportion of deaths from cardiovascular and cerebrovascular diseases represented by hypertension, etc., will increase from 28.8% in 1997 to 36.0% in 2002.

At present, there are six main types of drugs for the treatment of hypertension: diuretic antihypertensive drugs, β-blockers, calcium antagonists, angiotensin converting enzyme inhibitors (ACEI), and angiotensin II receptors. Antagonists (ARB) and alpha 1-receptor blockers. These drugs have achieved good results in the treatment of hypertension. Angiotensin-converting enzyme inhibitors are the first line of antihypertensive drugs with all six strong indications (heart failure, myocardial infarction, high risk factors for coronary heart disease, diabetes, chronic kidney disease, prevention of stroke recurrence). ACEI has been affirmed in the role of renal hypertension, chronic glomerulonephritis, chronic renal failure, and treatment for the prevention of diabetic nephropathy.

An ACE inhibiting peptide refers to a class of polypeptides having ACE inhibitory activity. These polypeptides differ in amino acid sequence and peptide chain length, but all have similar activities. ACE has two active sites, designated N-zone and C-region, respectively, which have similar functions but differ in affinity for different substrates. The binding of ACE inhibitory peptide to ACE inhibits angiotensin I, thereby preventing ACE from catalyzing the hydrolysis of angiotensin I to angiotensin II, thereby lowering blood pressure. At present, many studies have shown that some short peptides in food have hypotensive effects. ACE inhibitory peptides have been found in many food source materials. From the source, food source ACE inhibitory peptides can be divided into three categories: peptides from milk proteins, including cheese protein, lactoglobulin and whey protein; peptides from plant proteins, such as rice peptides, soy peptides, corn peptides Etc.; peptides derived from fish and shellfish, such as tilapia polypeptide, squid polypeptide, tuna polypeptide, sardine polypeptide, Antarctic krill polypeptide, and the like. Although the antihypertensive effects of these ACE inhibiting peptides are not as strong as synthetic drugs, their safety, non-toxic side effects have attracted many researchers. The short-chain ACE inhibitory peptides found in natural food proteins have not only demonstrated strong inhibitory activity against ACE in vitro, but also the blood pressure lowering effect in animal experiments and human clinical experiments. This provides a new way to prevent and relieve high blood pressure through functional foods.

Hairtail is mainly distributed in the north and northwest of the Pacific Ocean and China's East China Sea and Yellow Sea, with an annual catch of 5 million tons. The fish has low fat content, high nutritional value, and the meat is tender and delicious, and is very popular among people from different regions. The octopus is usually processed in the form of freezing or canning. During the processing, a large amount of fish head, internal organs and other scraps are produced, which account for about the weight of the raw fish. 40%~50%. These scraps are rich in protein resources. If they are not treated effectively, they will waste resources and cause environmental pollution.

Summary of the invention

It is an object of the present invention to provide a method for preparing an ACE inhibiting peptide using a fish waste.

The technical solution adopted by the present invention is:

A method for producing an ACE inhibiting peptide using a fish waste, comprising the following steps:

1) Wash the fish waste, add water, homogenate treatment, heat the enzyme to inactivate, to obtain a slurry A;

2) cooling the slurry A to not higher than 60 ° C, adding protease to enzymatic hydrolysis to obtain an enzymatic hydrolysate;

3) Centrifuging the enzymatic solution and collecting the supernatant A;

4) Add calcium carbonate to the supernatant A, mix and filter to obtain a clear supernatant B;

5) Ultrafiltration was carried out using a hollow fiber ultrafiltration device having a molecular weight cut off of 3000 Da, and the filtrate was collected and dried to obtain an ACE inhibitory peptide.

As a further optimization of the invention, the supernatant A is heated to inactivate the protease therein.

As a further optimization of the present invention, the dried ACE inhibiting peptide is further passed through Sephadex G-15 and RP-HPLC were separated and purified in two steps to obtain a high purity ACE inhibiting peptide.

As a further optimization of the present invention, the homogenate treatment is followed by heating to 90 ° C for at least 10 min to inactivate the enzyme.

As a further optimization of the present invention, the slurry A is cooled to 45 to 55 ° C, its pH is adjusted to 7.5 to 8.5, and then protease digestion is added.

As a further optimization of the present invention, the mass ratio of the fish waste to water is 1:3.

As a further optimization of the present invention, calcium carbonate is added in an amount of 10% by mass of the supernatant A.

As a further optimization of the present invention, the enzymatic hydrolysis time of the protease is 120 to 240 min.

The beneficial effects of the invention are:

The method of the invention can convert low-value squid scrap into high-value ACE inhibitory peptide, turn waste into treasure, and make full use of resources.

The method of the invention has simple and simple process route, simple operation, the equipment used is mature equipment, the investment cost is low, and the recovery rate of the ACE inhibiting peptide is high.

The high-purity ACE inhibitory peptide was isolated and purified by two steps of Sephadex G-15 and RP-HPLC, and had excellent biological activity, and its IC ₅₀ value for inhibiting ACE was 11.5 μM.

detailed description

A method for producing an ACE inhibiting peptide using a fish waste, comprising the following steps:

1) Wash the fish waste, add water, homogenate treatment, heat the enzyme to inactivate, to obtain a slurry A;

2) cooling the slurry A to not higher than 60 ° C, adding protease to enzymatic hydrolysis to obtain an enzymatic hydrolysate;

3) Centrifuging the enzymatic solution and collecting the supernatant A;

4) Add calcium carbonate to the supernatant A, mix and filter to obtain a clear supernatant B;

5) Ultrafiltration was carried out using a hollow fiber ultrafiltration device having a molecular weight cut off of 3000 Da, and the filtrate was collected and dried to obtain an ACE inhibitory peptide.

As a further optimization of the invention, the supernatant A is heated to inactivate the protease therein. Of course, the enzymes therein can also be inactivated by other known methods as needed.

As a further optimization of the present invention, the dried ACE inhibiting peptide is further passed through Sephadex G-15 and RP-HPLC were separated and purified in two steps to obtain a high purity ACE inhibiting peptide.

As a further optimization of the present invention, the homogenization treatment is followed by heating to 90 ° C to keep at least 10 Min to inactivate the enzyme. Of course, the enzymes therein can also be inactivated by other known methods as needed.

As a further optimization of the present invention, the slurry A is cooled to 45 to 55 ° C, its pH is adjusted to 7.5 to 8.5, and then protease digestion is added.

As a further optimization of the present invention, the squid scrap is mixed with water for the purpose of facilitating homogenization damage, and the mass ratio can be adjusted as needed. In particular, when the mass ratio is 1:3, the amount of water can be effectively reduced. At the same time, the adverse effects on the preparation of the peptide are also relatively small.

As a further optimization of the present invention, calcium carbonate is added in an amount of 10% by mass of the supernatant A.

As a further optimization of the present invention, the enzymatic hydrolysis time of the protease is 120-240 Min. Of course, depending on the enzyme used and the temperature of the enzymatic hydrolysis, appropriate shortening or extension can also be carried out.

The invention will now be further described in conjunction with the examples.

Example 1 Enzymatic hydrolysis of fish waste and enrichment of ACE inhibitory peptide

1) Processing of raw materials: Weigh 100 kg of fish waste, and after washing, add 300 L deionized water, after beating in the beater, boil for 20 min, inactivate the self-enzyme system;

2) Enzymatic digestion of fish waste: to be taken with the fish waste slurry, cooled to 55 ° C, adjusted to pH 8.0, added to alkaline protease Alcalase 150 g, enzymatic hydrolysis for 3 hr, to obtain an enzymatic hydrolysate;

3) Inactivate the alkaline protease Alcalase: heat the enzymatic hydrolyzate to 95 ° C, keep warm 10 Min, inactivated alkaline protease Alcalase in the enzymatic hydrolysate;

4) Pretreatment of the enzymatic hydrolysate: The enzymatic hydrolysate after the enzyme is cooled to room temperature, using a tube centrifuge at 3000 rpm Centrifuge down and collect the supernatant;

5) Clarification of the enzymatic hydrolysate: 10% light calcium carbonate was added to the supernatant collected by centrifugation, and the plate was filtered to obtain a clear, ACE-inhibiting peptide-containing serum;

6) Removal of macromolecular substances: using a hollow fiber ultrafiltration device with a molecular weight cut-off of 3000 Da, ultrafiltration to remove macromolecular substances in the supernatant containing the ACE inhibitory peptide, and collecting the filtrate containing the ACE inhibiting peptide;

7) Drying of ACE inhibiting peptide: The filtrate was spray-dried to obtain an ACE inhibiting peptide.

Example 2 Preparation of High Purity ACE Inhibitory Peptide

Separation of crude ACE inhibitor peptide powder by Sephadex G-15 ACE inhibitor peptide, gel column, 1.8×60 Cm; sample concentration, 50 mg/ml; sample loading, 1.5 ml; flow rate, 0.4 ml/min; eluent, 0.02 mol/L HAc-NaAc buffer (pH 4.0); obtaining 7 components, wherein the activity of component E is higher;

Table 1 ACE inhibitory activity of components obtained by Sephadex G-15 separation of enzymatic hydrolysate

Component	ACE inhibition rate (%)
Peak A	43.55±1.25
Peak B	73.48±2.10
Peak C	77.37±1.44
Peak D	50.68±1.67
Peak E	87.36±0.94
Peak F	36.65±1.52
Peak G	20.34±1.28

The component E was purified by RP-HPLC, and 16 fractions were separated by Kromasil C18 12 μm ST 4.6/250 (4.6×250 mm) column. The component F10 has high ACE inhibitory activity and was purified. The single active component F10 was analyzed and identified by amino acid composition, and a novel ACE inhibitory peptide LPP was isolated from the enzymatic hydrolyzed Alcalase, with an IC ₅₀ value of 11.5 μM.

Claims (9)

1.  A method for producing an ACE inhibiting peptide using a fish waste, comprising the following steps:

   1) Wash the fish waste, add water, homogenate treatment, heat the enzyme to inactivate, to obtain a slurry A;

   2) cooling the slurry A to not higher than 60 ° C, adding protease to enzymatic hydrolysis to obtain an enzymatic hydrolysate;

   3) Centrifuging the enzymatic solution and collecting the supernatant A;

   4) Add calcium carbonate to the supernatant A, mix and filter to obtain a clear supernatant B;

   5) Ultrafiltration was carried out using a hollow fiber ultrafiltration device having a molecular weight cut off of 3000 Da, and the filtrate was collected and dried to obtain an ACE inhibitory peptide.
2. The method of claim 1 wherein the supernatant A is heated to inactivate the protease therein.
3. The method according to claim 1 or 2, characterized in that the homogenization treatment is followed by heating to 90 ° C to keep at least 10 Min to inactivate the enzyme.
4. The method according to any one of claims 1 to 3, characterized in that the slurry A is cooled to 45 to 55 ° C, the pH thereof is adjusted to 7.5 to 8.5, and then protease digestion is added.
5. The method according to any one of claims 1 to 4, characterized in that the mass ratio of the squid scrap to water is 1:3.
6. The method according to any one of claims 1 to 5, characterized in that the protease is an alkaline protease Alcalase.
7. The method according to any one of claims 1 to 6, wherein the calcium carbonate is added in an amount of 10% by mass of the supernatant A.
8. The method according to any one of claims 1 to 7, wherein the enzymatic hydrolysis time of the protease is 120 to 240 min.
9. The method according to any one of claims 1 to 8, wherein the dried ACE inhibiting peptide is further passed through Sephadex G-15 and RP-HPLC were separated and purified in two steps to obtain a high purity ACE inhibiting peptide.