CN102191306A - Enzymatic preparation method for antihypertensive peptides from jellyfish - Google Patents

Abstract

The invention relates to an enzymatic preparation method of jellyfish antihypertensive peptide. It selects fresh jellyfish or alum-made jellyfish as raw materials, grinds them after desalination and washing; adjusts the protein concentration of the jellyfish pulp to 1% to 3%; adjusts the pH value to 2 to 3.5, and controls the temperature to 35 to 40°C. Add pepsin to keep warm for 4-6 hours, and the dosage of pepsin is 2000-4000u per gram of protein; then adjust the pH value of jellyfish pepsin hydrolyzate to 5-6, control the temperature at 50-55°C, and add papain for enzymolysis 1 to 3 hours, the dosage of papain is 2000 to 4000u per gram of protein; the enzyme is inactivated; then the jellyfish antihypertensive peptide powder is obtained through membrane filtration to remove impurities, nanofiltration membrane desalination, concentration, spray drying or freeze drying. The jellyfish antihypertensive peptide prepared by the invention is proved to have good auxiliary effect of lowering blood pressure through oral experiments on SHR rats (essentially hypertensive rats). The invention has advanced production technology, strong operability, complete protein enzymolysis and controllable product quality.

Description

Enzymatic preparation method of jellyfish antihypertensive peptide

technical field

The invention relates to a general preparation method of peptides, in particular to an enzymatic preparation method of jellyfish antihypertensive peptide obtained by enzymatically hydrolyzing jellyfish collagen peptide liquid, and then separating, concentrating and drying to obtain jellyfish antihypertensive peptide powder.

Background technique

Jellyfish, also known as jellyfish, is a marine coelomate. It not only has many types and large numbers, but also has a wide distribution. It is an important part of marine zooplankton communities. Jellyfish is extremely nutritious, and its body contains high protein and inorganic salts such as calcium, phosphorus, iodine, iron, etc., and is known as the "treasure of aquatic products". Jellyfish is not only edible, but also a good medicine for curing diseases blindly. According to "Compendium of Materia Medica": jellyfish has the functions of clearing away heat and detoxification, reducing phlegm and softening hardness, reducing blood pressure and reducing swelling, and has curative effects on bronchitis, asthma, hypertension, gastric ulcer and other diseases. Therefore, jellyfish, as a large organism with the same source of medicine and food, has important economic and food value.

The methods for preparing active peptides mainly include: direct extraction, in vitro degradation and synthesis of active peptides by using recombinant DNA technology, enzymatic method or chemical method. There are three technological routes for preparing active peptides by hydrolyzing proteins in vitro: acid hydrolysis, alkali hydrolysis and enzymatic hydrolysis. The former two methods are simple, but there are harmful effects in nutrition and toxicity, so their application in food processing is greatly limited. Enzymatic hydrolysis of protein generally does not cause nutritional loss, nor does it cause toxicological problems; at the same time, the enzyme is specific, can be carried out under mild conditions, and the energy consumption is also low, so enzymatic hydrolysis is widely used in food Often used in processing.

However, the technology of preparing jellyfish active peptides by enzymatically hydrolyzing jellyfish proteins has been reported, for example: "a kind of jellyfish active peptide" disclosed in the CN101422195A Chinese invention patent application published by the applicant and disclosed on May 6, 2009 Peptide yoghurt and its preparation method", CN101422266A Chinese invention patent application published on May 6, 2009, "a jellyfish nutrient solution and its preparation method" disclosed in the publication specification of CN101422266A Chinese invention patent application published on March 17, 2010, CN101669659A Chinese invention patent published on March 17, 2010 "A jellyfish and water chestnut drink and its preparation method" disclosed in the application publication specification, "a compound jellyfish antihypertensive granule and its preparation method" disclosed in the publication specification of CN101862375A Chinese invention patent application published on October 20, 2010 , in the above public documents, jellyfish raw materials are desalted, washed, crushed, diluted with water or enzymatically hydrolyzed with alkaline protease or trypsin to obtain jellyfish polypeptide liquid, which is then used to prepare other functional products. In the disclosed technology, a single enzyme is used for enzymatic hydrolysis of jellyfish. In protein hydrolysis, the choice of enzyme is the key technology. Different enzymes and separation and purification methods can be used to obtain different products. The activity of peptide products obtained by different enzymatic hydrolysis processes is different. At present, there is no report on the preparation of jellyfish antihypertensive peptide by double-enzyme hydrolysis.

Contents of the invention

In order to overcome the deficiencies in the prior art, the invention provides an enzymatic preparation method of jellyfish antihypertensive peptide with advanced production technology, controllable product quality, complete enzymatic hydrolysis of protein, and high activity of the obtained jellyfish peptide.

The technical scheme adopted by the present invention to solve the technical problem is: an enzymatic preparation method of jellyfish antihypertensive peptide, characterized in that: through the following steps

(a) Processing of raw materials Select fresh jellyfish or alum-made jellyfish as raw materials, soak in water to desalinate until the salt content is less than 5%, and use a colloid mill to grind after washing, so that the particle size of the obtained jellyfish pulp is controlled at 2-5um;

(b) The first step of enzymatic hydrolysis to measure the protein content of the jellyfish pulp obtained in step (a), add water to adjust the protein mass concentration to 1% to 3%; adjust the pH value to 2 to 3.5, control the temperature to 35 to 40°C, add Pepsin, the dosage of pepsin is to add 2000-4000u of pepsin per gram of protein, and keep it warm for 4-6 hours to get jellyfish pepsin hydrolyzate;

(c) The second step of enzymatic hydrolysis Adjust the pH value of the jellyfish pepsin hydrolyzate obtained in step (b) to 5-6, control the temperature at 50-55 ° C, add papain, the amount of papain is 2000-2000 per gram of protein 4000u of papain, hydrolyzed for 1 to 3 hours to get jellyfish dual-enzyme hydrolyzate;

(d) Enzyme inactivation Heat the jellyfish double-enzyme hydrolyzate obtained in step (c) to 90-100°C, keep it for 10-20 minutes, and perform enzyme inactivation to obtain the enzymatically inactivated jellyfish double-enzyme hydrolyzate;

(e) Removal of impurities and desalination The enzyme-inactivated jellyfish double-enzyme hydrolyzate obtained in step (d) is passed through a tubular membrane to remove impurities, and then desalted with a nanofiltration membrane with a molecular mass cut-off of 150-300, and concentrated to obtain jellyfish antihypertensive Peptide concentrate, the jellyfish antihypertensive peptide concentrate has a soluble solid content of 15% to 20%;

(f) Drying The jellyfish antihypertensive peptide concentrate obtained in step (e) is spray-dried or freeze-dried to obtain jellyfish antihypertensive peptide powder.

The jellyfish dual-enzyme enzymatic hydrolysis solution after the enzymatic liquid has been extinguished is described as a tubular membrane for removing impurities. Membrane pressure is controlled at 0.8-1.0 bar.

In the spray drying of the jellyfish antihypertensive peptide concentrate, the air inlet temperature is controlled at 185°C-190°C, the air outlet temperature is controlled at 75-80°C, and the spray pressure is 0.4MPa.

For the freeze-drying of the jellyfish antihypertensive peptide concentrate, the vacuum degree is controlled at 50-133Pa, and the heating plate temperature is: 20°C 250min/40°C 400min/30°C until the freeze-dried material has a constant weight;

The temperature of the cold trap is controlled at -40°C to -50°C.

The present invention uses fresh jellyfish or alum-made jellyfish as raw materials, after desalination and grinding, pepsin and papain are enzymatically hydrolyzed to obtain jellyfish proteolytic peptide liquid, and then the jellyfish is obtained by membrane filtration to remove impurities and nanofiltration membrane desalination and separation The antihypertensive peptide solution is concentrated, spray-dried or freeze-dried to obtain jellyfish antihypertensive peptide powder. The jellyfish antihypertensive peptide prepared by the invention is proved to have good auxiliary effect of lowering blood pressure through oral experiments on SHR rats (essentially hypertensive rats). The production technology of the jellyfish antihypertensive peptide is advanced, the operability is strong, the product quality is controllable, and the protein is completely enzymatically hydrolyzed. The jellyfish antihypertensive peptide powder obtained is white or light yellow, has high activity, is easily soluble in water, and can be used in food and medicine. fields such as healthcare.

Detailed ways

The present invention will be described in further detail below in conjunction with the examples.

Example 1

(a) Processing of raw materials Weigh 500kg of fresh jellyfish as raw material, soak in clear water to desalinate until the salt content is 3%, after cleaning, use a colloid mill for grinding treatment, and the particle size of the obtained jellyfish pulp is controlled at 3.5um;

(b) The first step of enzymatic hydrolysis Measure the water and protein content of the jellyfish pulp obtained in step (a). According to the measurement results, add water to adjust the protein mass concentration to 2%; add acid to adjust the pH value to 3.26, and control the temperature to 37 ° C. , add pepsin, the dosage of pepsin is to add 3000u pepsin per gram of protein, keep warm for 4 hours, and get jellyfish pepsin hydrolyzate;

(c) The second step of enzymatic hydrolysis Adjust the pH value of the jellyfish pepsin hydrolyzate obtained in step (b) to 5.02 with an alkaline solution, raise the temperature to 50°C, add papain, the amount of papain is 4000u per gram of protein Papain, enzymatically hydrolyzed for 1 hour, to obtain jellyfish dual-enzyme hydrolyzate;

(d) Enzyme inactivation Heat the jellyfish double-enzyme hydrolyzate obtained in step (c) to 95°C, keep it for 15 minutes, and perform enzyme inactivation to obtain the enzymatically inactivated jellyfish double-enzyme hydrolyzate;

(e) Removal of impurities and desalination The enzyme-inactivated jellyfish double-enzyme hydrolyzate obtained in step (d) is passed through a tubular membrane to remove impurities, wherein the working pore diameter of the tubular membrane is 0.15 μm, the working temperature is controlled at 35°C, and the membrane inlet pressure is controlled at 2.5bar, the membrane outlet pressure is controlled at 0.9bar. Then use a nanofiltration membrane with a molecular weight cut-off of 250 for desalting, and concentrate while desalting, and the obtained concentrated solution can be further concentrated in a vacuum to obtain a jellyfish antihypertensive peptide concentrated solution. The soluble solid content of the jellyfish antihypertensive peptide concentrated solution is 20%;

(f) Drying The jellyfish antihypertensive peptide concentrate obtained in step (e) is dried by freeze-drying. The working conditions of freeze-drying are: vacuum degree is controlled at 100Pa, heating plate temperature: 20°C 250min/40°C 400min/ 30°C until the freeze-dried material has constant weight; the temperature of the cold trap is controlled at -45°C. 3.5 kg of jellyfish antihypertensive peptide powder was obtained, and its IC ₅₀ was 0.4 mg/mL according to the determination of ACE inhibition rate in vitro.

Example 2

(a) Processing of raw materials Weigh 500kg of alum-made jellyfish as raw material, soak in clean water, rehydrate, desalinate until the salt content is less than 5%, clean it with running water, and use a colloid mill to grind it. The particle size of the obtained jellyfish pulp is controlled at 2um ;

(b) The first step of enzymatic hydrolysis Measure the water and protein content of the jellyfish pulp obtained in step (a). According to the measurement results, add water to adjust the protein mass concentration to 3%; add acid to adjust the pH value to 2, and control the temperature to 35°C , add pepsin, the dosage of pepsin is to add 4000u of pepsin per gram of protein, heat preservation enzymolysis for 5h, get jellyfish pepsin hydrolyzate;

(c) The second step of enzymatic hydrolysis Adjust the pH of the jellyfish pepsin hydrolyzate obtained in step (b) to 6 with an alkaline solution, control the temperature at 55°C, add papain, the amount of papain is 3500u of papaya per gram of protein Protease, enzymatic hydrolysis for 2 hours, to obtain jellyfish dual-enzyme hydrolyzate;

(d) Enzyme inactivation Heat the jellyfish double-enzyme hydrolyzate obtained in step (c) to 100°C, keep it for 10 minutes, and perform enzyme inactivation to obtain the enzymatically inactivated jellyfish double-enzyme hydrolyzate;

(e) Removal of impurities and desalination The enzyme-inactivated jellyfish double-enzyme hydrolyzate obtained in step (d) is passed through a tubular membrane to remove impurities, wherein the working pore size of the tubular membrane is 0.2 μm, the working temperature is controlled at 30°C, and the membrane inlet pressure is controlled. At 3bar, the exit membrane pressure is controlled at 1.0bar. Then use a nanofiltration membrane with a molecular weight cut-off of 300 for desalting, and concentrate while desalting, and the obtained concentrate is further concentrated by vacuum concentration to obtain a jellyfish antihypertensive peptide concentrated solution. The soluble solid content of the jellyfish antihypertensive peptide concentrated solution 15%;

(f) Drying The jellyfish antihypertensive peptide concentrated solution obtained in step (e) is spray-dried. During spray drying, the air inlet temperature is controlled at 190° C., the air outlet temperature is controlled at 75° C., and the spray pressure is 0.4 MPa to obtain jellyfish antihypertensive peptide. Pressed peptide powder 4.0kg, according to the determination of ACE inhibition rate in vitro, its _IC50 is 0.5mg/mL.

Example 3

(a) Processing of raw materials Weigh 300kg of jellyfish made from alum as raw material, soak in water, rehydrate, desalinate until the salt content is less than 4%, wash with running water, and use a colloid mill for grinding, and the particle size of the obtained jellyfish pulp is controlled at 5um;

(b) The first step of enzymatic hydrolysis to determine the moisture and protein content of the jellyfish pulp obtained in step (a), adding water to adjust the protein mass concentration to 1%; adding acid to adjust the pH value to 3, controlling the temperature to 40°C, adding pepsin, The dosage of pepsin is to add 2000u of pepsin per gram of protein, and keep it warm for 6 hours to get jellyfish pepsin hydrolyzate;

(c) The second step of enzymatic hydrolysis Adjust the pH value of the jellyfish pepsin hydrolyzate obtained in step (b) to 5.5 with alkaline solution, control the temperature at 52°C, add papain, the amount of papain is 2000u of papaya per gram of protein Protease, enzymatic hydrolysis for 3 hours, to obtain jellyfish dual-enzyme hydrolyzate;

(d) Enzyme inactivation Heat the jellyfish double-enzyme hydrolyzate obtained in step (c) to 90°C, keep it for 20 minutes, and perform enzyme inactivation to obtain the enzymatically inactivated jellyfish double-enzyme hydrolyzate;

(e) Impurity removal and desalination The enzyme-inactivated jellyfish double-enzyme hydrolyzate obtained in step (d) is passed through a tubular membrane to remove impurities. The working pore size of the tubular membrane is 0.1 μm, the working temperature is controlled at 40 ° C, and the membrane inlet pressure is controlled. At 2.8bar, the exit membrane pressure is controlled at 0.8bar. Then use a nanofiltration membrane with a molecular weight cut-off of 150 for desalting, and concentrate while desalting, and the obtained concentrated solution is further concentrated by vacuum concentration to obtain a jellyfish antihypertensive peptide concentrated solution. The soluble solid content of the jellyfish antihypertensive peptide concentrated solution is 18%;

(f) Drying The jellyfish antihypertensive peptide concentrate obtained in step (e) is freeze-dried. The working conditions of freeze-drying are: vacuum degree is controlled at 133Pa, heating plate temperature: 20°C 250min/40°C 400min/30°C until frozen The weight of the dry material was constant; the temperature of the cold trap was controlled at -50°C, and 2.5 kg of jellyfish antihypertensive peptide powder was obtained. According to the determination of ACE inhibition rate in vitro, its IC ₅₀ was 0.4 mg/mL.

The SHR rat model was used to evaluate the prepared jellyfish antihypertensive peptide powder, and the experimental results are shown in Table 1.

mmHg)Table 1 The effect of each administration group on SHR tail artery systolic pressure (

mmHg)

Note: Compared with blank control group, *P<0.05, **P<0.01

Compared with the blank control group, the blood pressure levels of the rats in the jellyfish antihypertensive peptide group were significantly lowered after 4 weeks of intragastric administration (P<0.05), and the administration group had no significant effect on the body weight of the rats. Jellyfish antihypertensive peptide can significantly reduce the blood pressure of essential hypertensive rats.

Claims (4)

1. the enzymatic-process preparation method of a jellyfish step-down peptide is characterized in that: through the following step

(a) processing of raw material is chosen fresh jellyfish or alum system jellyfish is a raw material, and clear water soaks desalination to salinity＜5%, cleans the back and adopts colloidal mill to carry out milled processed, and gained jellyfish slurry particle fineness is controlled at 2～5um;

(b) protein content of the first step enzymolysis determination step (a) gained jellyfish slurry, adding water, to adjust proteinic mass concentration be 1%～3%; Adjusting the pH value is 2～3.5, and controlled temperature is 35～40 ℃, adds stomach en-, and pepsic consumption is the stomach en-that every gram protein adds 2000～4000u, and insulation enzymolysis 4～6h gets jellyfish pepsin hydrolysis liquid;

(c) the second step enzymolysis is 5～6 with the jellyfish pepsin hydrolysis liquid adjustment pH value that step (b) obtains, controlled temperature is 50～55 ℃, adds papoid, and the papoid consumption is the papoid that every gram protein adds 2000～4000u, enzymolysis 1～3h gets the two enzyme enzymolysis solutions of jellyfish;

(d) enzyme-deactivating is heated to 90～100 ℃ with the two enzyme enzymolysis solutions of jellyfish that step (c) obtains, and keeps 10～20 minutes, carries out enzyme-deactivating, the two enzyme enzymolysis solutions of the enzyme jellyfish of must going out;

(e) impurity elimination, desalination are removed impurity with the two enzyme enzymolysis solutions of the enzyme jellyfish of going out that step (d) obtains through tubular membrane, adopt the nanofiltration membrane desalination of 150～300 trapped molecular weights then, and concentrate, get jellyfish step-down peptide concentrated solution, the soluble solid content of this jellyfish step-down peptide concentrated solution is 15%～20%;

(f) the dry jellyfish step-down peptide concentrated solution that step (e) is obtained carries out spraying drying or lyophilize, obtains jellyfish step-down peptide powder.

2. the enzymatic-process preparation method of a kind of jellyfish step-down peptide according to claim 1, it is characterized in that: the two enzyme enzymolysis solutions of described jellyfish after enzyme liquid is gone out are removed the tubular membrane of impurity, its work aperture is 0.1～0.2 μ m, working temperature is controlled at 30～40 ℃, advance film pressure and be controlled at 2.5～3bar, the membrane pressure-controlling is at 0.8～1.0bar.

3. the enzymatic-process preparation method of a kind of jellyfish step-down peptide according to claim 1 and 2, it is characterized in that: the described spraying drying that jellyfish step-down peptide concentrated solution is carried out, its inlet temperature is controlled at 185 ℃～190 ℃, and air outlet temperature is controlled at 75～80 ℃, and spray pressure is 0.4MPa.

4. the enzymatic-process preparation method of a kind of jellyfish step-down peptide according to claim 1 and 2, it is characterized in that: the described lyophilize that jellyfish step-down peptide concentrated solution is carried out, its vacuum degree control is at 50～133Pa, and temperature of heating plate: 20 ℃ 250min/40 ℃ 400min/30 ℃ up to the freeze-dried material constant weight; Condenser temperature is controlled at-40 ℃～-50 ℃.