CN114507702B - Marine antarctic krill peptide and application thereof - Google Patents
Marine antarctic krill peptide and application thereof Download PDFInfo
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- CN114507702B CN114507702B CN202210115111.1A CN202210115111A CN114507702B CN 114507702 B CN114507702 B CN 114507702B CN 202210115111 A CN202210115111 A CN 202210115111A CN 114507702 B CN114507702 B CN 114507702B
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- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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Abstract
The invention relates to the technical field of functional polypeptide preparation, in particular to ocean antarctic krill peptide and application thereof; the euphausia superba proteolytic peptide is prepared by taking fresh euphausia superba or frozen euphausia superba as raw materials, crushing, and respectively carrying out enzymolysis by trypsin, pepsin and papain. The polypeptide prepared by the invention has the effect of reducing cholesterol, thereby improving the application and development values of the antarctic krill.
Description
Technical Field
The invention belongs to the technical field of functional polypeptide preparation, and particularly relates to ocean antarctic krill peptide and application thereof.
Background
Euphausia superba (Euphausia superba) is a shrimp of the genus Euphausia of the family Euphausiaceae. Euphausia superba has typical high protein, low fat characteristics with protein and fat contents of 16.31% and 1.3%, respectively. The krill has rich mineral content, the mineral content is 2.76 percent, which is higher than that of Penaeus japonicus (1.6 percent), clams (2.2 percent) and other marine products. The protein hydrolysate of antarctic krill contains 18 amino acids, including 8 essential amino acids required by human body. Wherein the glutamic acid content is highest and the lysine is next to the highest. The lipid of Euphausia superba has the highest polar lipid content, and the secondary triglyceride content is 56-81% and 12-38% of total fat content respectively. Euphausia superba also contains various active substances such as protein digestive enzymes, cytokinin amino acids, etc. Therefore, the antarctic krill has important economic and edible values.
The method for obtaining the marine active polypeptide comprises direct extraction, in vitro degradation, fermentation of engineering bacteria, enzymatic hydrolysis or chemical synthesis and the like. The enzymatic hydrolysis of protein does not cause nutrition loss and toxicological problems; meanwhile, the enzyme action has specificity, high efficiency and specificity, can be performed under mild conditions and has low energy consumption, so that the enzymatic hydrolysis is widely applied to food processing.
Technology for preparing antarctic krill active peptides by enzymatic hydrolysis of antarctic krill proteins has been reported. For example, the preparation and use of a protein hydrolysate of ice fresh antarctic krill is disclosed in chinese patent application No. CN201410075726.1, wherein the invention discloses crushing the raw materials of antarctic krill, obtaining an enzymatic hydrolysate by alkaline protease, then carrying out solid-liquid separation and drying to obtain antarctic krill peptide powder, thereby further processing and preparing other functional products. However, the enzymolysis of the euphausia superba is carried out by adopting single enzyme, and the euphausia superba peptide powder is obtained by direct solid-liquid separation and drying without further purification. In addition, in the preparation process of the active peptide, enzyme selection and enzymolysis liquid purification are core technologies, and the product activities of peptides obtained by different processes are different.
Disclosure of Invention
The invention provides ocean antarctic krill peptide and application thereof, and the prepared polypeptide has the effect of reducing cholesterol, so that the application and development value of antarctic krill is improved.
The invention provides a cholesterol-reducing antarctic krill peptide, which is prepared by taking fresh antarctic krill or frozen antarctic krill as a raw material, crushing, respectively carrying out enzymolysis step by using trypsin, pepsin and papain to obtain antarctic krill proteolytic peptide liquid, and carrying out spray drying or vacuum freeze drying;
further, the euphausia superba peptide is prepared by filtering euphausia superba protein hydrolysis peptide liquid through an ultrafiltration membrane with the molecular weight of 1000Da, and then spray drying or vacuum freeze drying the obtained filtrate;
further, the hydrolysis conditions of trypsin are as follows: the temperature is 50 ℃, the pH value is 7.0, and the enzymolysis time is 7 hours;
further, the hydrolysis conditions of the pepsin are as follows: the temperature is 50 ℃, the pH value is 7.0, and the enzymolysis time is 7 hours;
further, the papain hydrolysis conditions are as follows: the temperature is 55 ℃, the pH value is 7.0, and the enzymolysis time is 6 hours;
further, the amino acid sequences of the antarctic krill peptides are EIEVDENDEH, NTGDPVIREDE, SYVGQKDAQGED respectively.
The antarctic krill peptide provided by the invention is used for preparing products for reducing cholesterol;
the invention also provides a product with cholesterol reducing effect, which comprises the antarctic krill peptide with pharmacological effective concentration.
The cholesterol-lowering antarctic krill peptide prepared by the invention has better auxiliary cholesterol-lowering effect proved by a C57BL/6 hyperlipidemia model male SD rat lavage experiment. The production process of the cholesterol-reducing antarctic krill peptide has strong operability, controllable product quality and complete protein enzymolysis degree, and the obtained cholesterol-reducing antarctic krill peptide has high activity and can be used in the fields of food, medical care and the like.
Drawings
Fig. 1: an analytical identification chart of the P liquid component of the active peptide of the euphausia superba in the embodiment 3 of the invention;
fig. 2: an assay for triglycerides in rat serum in example 3 of the present invention;
fig. 3: a graph showing the measurement of total cholesterol in rat serum in example 3 of the present invention;
fig. 4: an assay for high density lipoprotein in rat serum in example 3 of the present invention;
fig. 5: a graph showing the measurement of low density lipoprotein in rat serum in example 3 of the present invention.
Detailed Description
The invention is described in further detail below in connection with examples.
Example 1 screening of preparation method of Euphausia superba peptide
1) Selection of protease: the hydrolysis degree is used as an index, and under the conditions that the enzyme addition amount is 100u/g, the feed-liquid ratio is 1:1g/ml and the enzymolysis time is 6 hours, the enzymolysis effects of six different proteases on euphausia superba proteins under the most proper enzymolysis conditions (see table 1) are compared. Finally pepsin, trypsin and papain were determined as the most suitable enzymes.
Table 1: information table of optimal enzymolysis temperature and pH value of different proteases
2) And analyzing the influence of temperature, pH value, feed-liquid ratio, enzyme addition amount and enzymolysis time on the prepared polypeptide by taking the hydrolysis degree as an index.
Adding water into euphausia superba to carry out homogenate to obtain euphausia superba homogenate, regulating the pH value corresponding to the selected protease, adding the protease, and carrying out enzymolysis for a certain time; after the reaction is finished, the enzymolysis liquid is placed in boiling water bath to be heated for inactivating protease, 10000g is centrifugated for 20min, and the supernatant polypeptide liquid is taken for hydrolysis degree measurement. And on the basis of a single factor experiment, carrying out response surface optimization analysis by using Design-expert8.0 software.
Table 2: papain enzymolysis process response surface experimental design factor level coding table
Table 3: response surface experimental scheme and result table of papain enzymolysis process
Table 4: trypsin enzymolysis process response surface experimental design factor horizontal coding table
Table 5: trypsin enzymolysis process response surface experimental scheme and result table
Table 6: pepsin enzymolysis process response surface experimental design factor level coding table
Table 7: pepsin enzymolysis process response surface experimental scheme and result table
The results show that the optimal enzymolysis process of papain is as follows: the temperature is 55 ℃, the pH value is 7.0, the feed-liquid ratio is 1.0g/ml, the enzyme addition amount is 100u/g, and the enzymolysis time is 6h; the optimal enzymolysis process of the trypsin is 50 ℃, the pH value is 7.0, the feed-liquid ratio is 1.54g/ml, the enzyme addition amount is 100u/g and the enzymolysis time is 7h, the optimal enzymolysis process of the pepsin is 50 ℃, the pH value is 7.0, the feed-liquid ratio is 1.58g/ml, the enzyme addition amount is 100u/g and the enzymolysis time is 7h.
Table 8: table of influence of different protease combinations on proteolytic effect of euphausia superba
Remarks: a-papain B-trypsin C-pepsin.
The best combination determined from the results of table 8 is as follows: firstly adding trypsin, wherein the temperature is 50 ℃, the pH value is 7.0, the feed-liquid ratio is 1.54g/ml, the enzyme adding amount is 100u/g, and the enzymolysis time is 7h; then adding pepsin, continuing to perform enzymolysis for 7 hours at the temperature of 50 ℃ and the pH value of 7.0, the feed-liquid ratio of 1.58g/ml and the enzyme addition amount of 100u/g, and finally adding papain, wherein the temperature of 55 ℃ and the pH value of 7.0, the feed-liquid ratio of 1.0g/ml, the enzyme addition amount of 100u/g and the enzymolysis time of 6 hours; the cholesterol reducing effect of the obtained hydrolysate can be reduced by 73.64%. The obtained antarctic krill peptide has the best cholesterol reducing effect under the enzymolysis condition.
Example 2: filtering and concentrating the prepared polypeptide
(1) And (3) raw material treatment: weighing 50kg of fresh shrimp meat of Euphausia superba as a raw material, cleaning with ultrapure water, putting the cleaned fresh shrimp meat into a pulverizer for pulverizing, and controlling the particle size of the Euphausia superba slurry to be 140 mu m;
(2) The first step of enzymolysis: measuring the moisture and protein content of the euphausia superba obtained in the step (1), and adding water to adjust the mass concentration of the protein to 3% according to the measurement result; adjusting the pH value to 7.0, controlling the temperature to 50 ℃, adding trypsin, wherein the dosage of trypsin is 100u of trypsin per gram of protein, and carrying out heat preservation and enzymolysis for 7 hours to obtain euphausia superba trypsin hydrolysate;
(3) And step two, enzymolysis: adjusting the pH value of the euphausia superba trypsin hydrolysate obtained in the step (2) to 7.0, keeping the temperature at 50 ℃, adding pepsin, and carrying out enzymolysis for 7 hours by adding 100u of pepsin into each gram of protein to obtain euphausia superba double-enzyme hydrolysate;
(4) And thirdly, enzymolysis: adjusting the pH value of the antarctic krill double-enzyme enzymolysis liquid obtained in the step (3) to 7.0, adjusting the temperature to 55 ℃, adding papain, wherein the dosage of the papain is 100u of papain per gram of protein, and carrying out enzymolysis for 4 hours to obtain the antarctic krill composite enzymolysis liquid;
(5) Separating and concentrating: centrifuging the euphausia superba compound enzymatic hydrolysate obtained in the step (4) for 20min at 10000g, and filtering the supernatant by using a 1000Da ultrafiltration membrane to obtain euphausia superba active peptide filtering concentrate with the molecular weight smaller than 1000 Da; and concentrated. The cholesterol reducing effect of the filtering concentrated solution is better than the effect of 500mg/kg of the antarctic krill compound enzymatic hydrolysate in the step (4) at the dosage of 300 mg/kg.
Example 3: purification of polypeptides
1) And (3) purifying: separating and purifying the antarctic krill active peptide concentrate prepared in the step 5) of the example 2 by using a SehadexG-25 gel column (1.6 cm multiplied by 30 cm), wherein the eluent is double distilled water, the elution speed is 0.5mL/min, and collecting the component with the highest activity;
2) And step two, purifying: the highest active fraction obtained in step 1) was further purified by separation using SP Sephadex C-25 cation exchange column (2.6 cm. Times.30 cm). Wherein the equilibrium solution is 20mM acetic acid buffer solution (pH=4.0), the acetic acid buffer solution containing NaCl is used for carrying out linear gradient elution, the elution speed is 0.5mL/min, and the component with highest activity is collected;
3) And step three, purifying: subjecting the component obtained in step 2) to reversed-phase high performance liquid chromatography C 18 The semi-preparative column (9.4 cm. Times.250 cm) was further separated and purified by gradient elution with 10-20% acetonitrile for 40min at a rate of 0.7mM/min at a column temperature of 30deg.C and collecting the main peak at 220nm to give Euphausia superba active peptide P solution (FIG. 1).
The P liquid component of the antarctic krill active peptide is analyzed and identified by an LC-MS technology, and 3 polypeptide sequences are obtained, and the information of the polypeptide sequences is shown in Table 9.
Table 9: polypeptide information table of antarctic krill active peptide P liquid
The p-1, p-2 and p-3 polypeptides are sent to biological companies for synthesis, and animal experiments are carried out on the synthesized polypeptides to verify the cholesterol reducing effect.
Establishing a hyperlipidemia animal model: the experiment adopts 200 SPF-grade male SD adult rats, the weight is about 91-112g, 30 SD rats are randomly selected as blank groups, the normal feed is fed, the rest SD rats are fed with D12492 high-fat feed, the model group is prepared, all animals drink water freely, 5 SD rats are randomly extracted from the blank groups and the model group respectively every week, blood is taken from tail veins, and TC (total cholesterol) and TG (triglyceride) are detected to judge whether the model is successful. After 6 weeks the molding animals had increased their TC levels approximately 2 times that of the blank, and were considered successful molding.
Cholesterol-lowering antarctic krill active peptide P animal experiments: 100 SD rats with successful model building are selected and randomly divided into 5 groups, namely an F1 group, an F2 group, an F3 group, an F4 group and an F5 group, and 20 SD rats in each group. Group F1 was subjected to gavage at a dose of 400mg/kg in an aqueous solution of the sample (euphausia superba active peptide filtered concentrate prepared in example 2); f2 group administration (p-1 synthetic polypeptide) 300mg/kg of the dose lavage; group F3 (p-2 synthetic polypeptide) 300mg/kg of the dose lavage; group F4 (p-3 synthetic polypeptide) 300mg/kg and group F5 (equal volume of 0.9% saline); each group was fed with D12492 high fat diet.
All SD rats were free to drink water, were perfused once daily, were continuously perfused for 8 weeks, were fed with 12h of weaning, and were blood-extracted from the eyeballs to prepare serum. Total Cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) in serum were measured, and the experimental results are shown in Table 10.
Table 10: SD rat serum index detection result table (n=20, mean.+ -. SD)
Note that: compared with the F5 group * P＜0.05, ** P＜0.01；
From Table 10 and FIGS. 2-5, after 8 weeks of gastric lavage of SD rats, the experimental rats of groups F1 to F4 had decreased in serum triglycerides, total cholesterol, high density lipoproteins and low density lipoproteins, which were significantly different from group F5 ** P < 0.01). The F2, F3 and F4 groups showed reduced levels of triglycerides, total cholesterol, high density lipoproteins and low density lipoproteins in the serum compared to the F1 group. The results show that the cholesterol lowering effect of the p-1, p-2 and p-3 polypeptides is better than the unpurified polypeptides of example 2.
Claims (2)
1. A marine antarctic krill peptide, wherein the amino acid sequence of the marine antarctic krill peptide is EIEVDENDEH, NTGDPVIREDE or SYVGQKDAQGED.
2. Use of the antarctic krill peptide of claim 1 for the preparation of a cholesterol-lowering product.
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