阅读说明：本技术 一种新型钙螯合肽及其制备方法和应用 (Novel calcium chelating peptide and preparation method and application thereof ) 是由 苗建银 廖婉雯 曹庸 于 2019-09-03 设计创作，主要内容包括：本发明涉及一种新型钙螯合肽及其制备方法和应用。所述钙螯合肽的氨基酸序列：YQEPVIAPKL。本发明提供的钙螯合肽安全无毒,与传统补钙剂相比具有更好的理化活性,其钙螯合能力达28.4mg/g；可在提高钙在人体中的吸收和生物利用率的同时,也可补充胶原蛋白肽,且可促进Caco-2小肠上皮细胞中钙吸收和转运,用作药物开发以及生物补钙剂的原料。本发明提供的制备方法成功得到该钙螯合肽,可充分利用鱼产品资源,操作简单,为罗非鱼骨的高值化利用提供了新途径。(The invention relates to a novel calcium chelating peptide and a preparation method and application thereof. The amino acid sequence of the calcium chelating peptide: YQEPVIAPKL are provided. The calcium chelating peptide provided by the invention is safe and nontoxic, has better physicochemical activity compared with the traditional calcium supplement, and has the calcium chelating capacity of 28.4 mg/g; can improve the absorption and bioavailability of calcium in human body, supplement collagen peptide, promote calcium absorption and transport in Caco-2 small intestine epithelial cells, and be used as raw material for drug development and biological calcium supplement. The preparation method provided by the invention successfully obtains the calcium chelating peptide, can fully utilize fish product resources, is simple to operate, and provides a new way for high-value utilization of tilapia mossambica bones.)

1. A novel calcium chelating peptide, wherein the amino acid sequence of the calcium chelating peptide is as follows: YQEPVIAPKL are provided.

2. The novel calcium chelating peptide of claim 1, wherein the molecular weight of the calcium chelating peptide is 1157.627 Da.

3. The method for producing the novel calcium chelating peptide according to any one of claims 1 to 2, which comprises the steps of:

s1: carrying out enzymatic hydrolysis on tilapia mossambica bone to remove meat, carrying out alkaline washing degreasing, decalcification treatment and acid treatment to obtain tilapia mossambica bone collagen;

s2: carrying out enzymolysis on tilapia mossambica bone collagen obtained in the step S1, inactivating enzyme, centrifuging, taking supernate and freeze-drying;

s3: and (3) performing multi-stage step-by-step separation and purification by using preparative reverse phase high performance liquid chromatography, and performing analytical high performance liquid separation to obtain the calcium chelating peptide.

4. The method of claim 3, wherein the enzymatic meat dissociation process in S1 is: cutting tilapia mossambica skeleton into segments, and removing minced meat on tilapia mossambica skeleton after enzymolysis by neutral protease.

5. The preparation method according to claim 3, wherein the alkaline cleaning degreasing process in S1 is as follows: and cleaning and degreasing by using an inorganic alkaline solution, and then cleaning to be neutral.

6. The method according to claim 3, wherein the decalcification treatment in S1 is carried out by: and (3) placing the tilapia mossambica bone degreased by alkali into an EDTA-2Na solution, freezing, stirring and cleaning to be neutral.

7. The method according to claim 3, wherein the acid treatment in S1 is carried out by: and (3) treating the decalcified fishbone with an acidic solution, and washing with water to be neutral to obtain tilapia mossambica bone collagen.

8. The method according to claim 3, wherein papain is selected for enzymolysis in S2;

the mass fraction of the papain in an enzymolysis system is 0.3-1%; the enzymolysis time is 4-6 h;

the temperature of enzymolysis is 40-60 ℃; the pH value of enzymolysis is 6.0-7.0, and the enzyme-substrate ratio of papain to tilapia bone collagen is 0.5-1.5%.

9. The method according to claim 3, wherein the preparative high performance liquid chromatography in S3 comprises 2-stage separation and purification; the conditions of the analytical high-performance liquid phase separation are as follows: the sample injection amount is 20 mu L; the chromatographic column is a C18 chromatographic column; mobile phase a was an aqueous solution containing 0.1% TFA, mobile phase B was an acetonitrile solution containing 0.1% TFA; the elution speed is 1.0 mL/min; the ultraviolet detection wavelength is double detection wavelength: 214nm and 280 nm; the elution conditions were: 0-40 min: mobile phase B: 5% -25%, 40-45 min: mobile phase B: 25% -90%, 45-50 min: mobile phase B: 90%, 50-55 min: mobile phase B: 5 percent.

10. Use of the novel calcium-chelating peptide according to any one of claims 1 to 2 for the preparation of a calcium supplement preparation or a functional beverage.

Technical Field

The invention belongs to the technical field of functional health food preparation, and particularly relates to novel calcium chelating peptide and a preparation method and application thereof.

Background

China is the largest tilapia culture and export country in the world. With the change in eating habits, consumers prefer to eat processed fish because of the greater convenience of eating. In 2017, the processing trade volume of the Chinese tilapia is about 67 ten thousand tons. Meat is a main product of tilapia, and tilapia bones become byproducts, which can pollute the environment if the tilapia bones cannot be effectively utilized. Research shows that 4.29 ten thousand tons of fishbone can be obtained from 30 ten thousand tons of tilapia. Fishbone is rich in collagen (about 40.8%) and calcium (2.72%), which are important sources of collagen and minerals. Calcium is an essential nutrient for the human body and plays an important role in bone growth, intracellular metabolism, muscle contraction, blood coagulation, skeletal structure support and the like.

Adequate calcium intake is essential to maintain bone density and provide maximum protection against negative calcium balance, especially for the elderly. The ingestion of calcium from the diet is the only source of calcium for the body to supplement bone. The amount of soluble calcium in the proximal duodenum and jejunum determines the amount of calcium absorbed by the body. However, it is affected by dietary factors such as phytic acid, oxalic acid and tannic acid which form calcium insoluble salts, as well as the alkaline pH of the intestinal tract. In recent years, the research shows that the peptide with certain characteristics in amino acid composition can chelate calcium and improve the bioavailability of the calcium. For example, peptides extracted from octopus fragments, casein, and fission yeast protein hydrolysates can chelate calcium, form peptide-calcium complexes, and enhance calcium absorption by Caco-2 cells.

In addition, studies report that calcium-chelating peptides can improve the absorption of calcium by rats, such as Pacific codfish bone collagen peptide, crucian carp skin collagen peptide, desalted duck egg white peptide and the like. Tilapia bone is an ideal raw material for producing bioactive peptide. Due to its low cost, the protein content is high. Extensive research has been carried out on the widespread utilization of fishbone, particularly on the utilization of fishbone calcium binding capacity. However, little research has been done on the bioavailability of monomeric peptides from tilapia bone.

Therefore, the method for developing the monomeric peptide with better bioavailability aiming at the tilapia mossambica bone has important research significance and economic value for realizing the further development of the tilapia mossambica bone.

Disclosure of Invention

The invention aims to overcome the defect or deficiency of the lack of the existing research on the bioavailability of tilapia mossambica bone monomeric peptide, and provides a novel calcium chelating peptide. The novel calcium chelating peptide provided by the invention has good calcium chelating activity, the calcium chelating capacity of the novel calcium chelating peptide reaches 28.4mg/g, the calcium transport in Caco-2 cell monolayers can be promoted, the novel calcium chelating peptide can be used for preparing calcium supplement preparations or functional beverages, the absorption and bioavailability of calcium in a human body are improved, meanwhile, the collagen peptide can be supplemented, and the requirement of the human body on the calcium element intake is met.

Another object of the present invention is to provide a method for producing the above-mentioned calcium chelating peptide.

The invention also aims to provide the application of the calcium chelating peptide in preparing calcium supplement preparations or functional beverages.

In order to achieve the purpose, the invention adopts the following technical scheme:

a calcium chelating peptide having the amino acid sequence: YQEPVIAPKL (Tyr-Gln-Glu-Pro-Val-Ile-Ala-Pro-Lys-Leu).

The calcium chelating peptide provided by the invention is safe and nontoxic, has better physicochemical activity compared with the traditional calcium supplement, has the calcium chelating capacity of 28.4mg/g, can promote calcium transport in Caco-2 cell monolayers, can supplement collagen peptide while improving the absorption and bioavailability of calcium in a human body, is used as a raw material for drug development and biological calcium supplement, and meets the requirement of the human body on the intake of calcium elements.

Preferably, the calcium chelating peptide has a molecular weight of 1157.627 Da.

The preparation method of the calcium chelating peptide comprises the following steps:

s1: carrying out enzymatic hydrolysis on tilapia mossambica bone to remove meat, carrying out alkaline washing degreasing, decalcification treatment and acid treatment to obtain tilapia mossambica bone collagen;

s2: carrying out enzymolysis on tilapia mossambica bone collagen obtained in the step S1, inactivating enzyme, centrifuging, taking supernate and freeze-drying;

s3: and (3) performing multi-stage step-by-step separation and purification by using preparative reverse phase high performance liquid chromatography, and performing analytical high performance liquid separation to obtain the calcium chelating peptide.

The invention takes calcium chelating activity as an evaluation index, and adopts preparative reverse phase high performance liquid chromatography and analytical high performance liquid chromatography to separate and purify tilapia mossambica bone collagen enzymolysis products, so as to obtain the tilapia mossambica bone collagen source calcium chelating peptide.

The preparation method provided by the invention successfully obtains the calcium chelating peptide, can fully utilize fish product resources, is simple to operate, and provides a new way for high-value utilization of tilapia mossambica bones.

Preferably, the step S1 further comprises the steps of thawing the tilapia mossambica skeleton, removing impurities, washing, and removing the head of the tilapia mossambica skeleton before the enzymatic degeeding.

Preferably, the enzymatic meat-removing process in S1 is: cutting tilapia mossambica skeleton into segments, and removing minced meat on tilapia mossambica skeleton after enzymolysis by neutral protease.

More preferably, the tilapia mossambica skeleton is cut into 4-7 cm sections in S1; the enzymolysis time of the neutral protease is 1-3 h, the temperature is 45-60 ℃, and the pH is 7-8.

More preferably, the mass concentration of the neutral protease is 5 g/mL; the mass ratio of the neutral protease to the tilapia mossambica skeleton is 1: 200; the enzymolysis time is 2h, the temperature is 55 ℃, and the pH is 7.5.

After the enzymolysis is finished, impurities are removed by washing.

Preferably, the alkaline degreasing process in S1 is as follows: and cleaning and degreasing by using an inorganic alkaline solution, and then cleaning to be neutral.

More preferably, the inorganic alkaline solution is a sodium hydroxide solution.

Specifically, the alkaline cleaning degreasing process in S1 is as follows: the fishbone is put into 0.1mol/L NaOH solution (w/v1:10), stirred for 24h and washed to be neutral.

Preferably, the decalcification process in S1 is as follows: and (3) placing the tilapia mossambica bone degreased by alkali into an EDTA-2Na solution, freezing, stirring and cleaning to be neutral.

More preferably, the pH value of the EDTA-2Na solution is 7.2, and the mass-to-volume ratio of the tilapia mossambica bone to the EDTA-2Na solution is 1: 10.

Specifically, the decalcification process comprises the following steps: the fishbone was placed in 10% EDTA-2Na (pH7.2, w/v1:10) and placed in a freezer at 4 ℃ while stirring for 5day, and then the fishbone was washed to neutrality for use.

Preferably, the acid treatment process in S1 is: and (3) treating the decalcified fishbone with an acidic solution, and washing with water to be neutral to obtain tilapia mossambica bone collagen.

More preferably, the acidic solution is a 4% hydrochloric acid solution; the acid treatment time was 18 h.

Preferably, papain is selected for enzymolysis in S2; the mass fraction of the papain in an enzymolysis system is 0.3-1%; the enzymolysis time is 3-6 h; the temperature of enzymolysis is 40-60 ℃; the pH value of the enzymolysis is 6-7.

More preferably, the mass fraction of the papain in the enzymolysis system is 1%; the enzymolysis time is 5 hours; the temperature of the enzymolysis is 60 ℃; the pH value of the enzymolysis is 7.0, and the enzyme substrate ratio of the papain to the tilapia bone collagen is 1%.

Preferably, the preparative high performance liquid chromatography described in S3 comprises two-stage stepwise separation and purification.

More preferably, the conditions of the first-stage separation by preparative high performance liquid chromatography are as follows: the sample injection amount is 4000 mu L; the chromatographic column is a C18 chromatographic column; mobile phase a was an aqueous solution containing 0.1% TFA, mobile phase B was an acetonitrile solution containing 0.1% TFA; the elution speed is 10.0 mL/min; the ultraviolet detection wavelength is double detection wavelength: 214nm and 280 nm; the elution conditions were: 0-10 min: mobile phase B: 6% -10%, 10-60 min: mobile phase B: 28%, 60-65 min: mobile phase B: 28% -90%, 65-75 min: mobile phase B: 90%, 75-80 min, mobile phase B: 90% -6%, 80-90 min: mobile phase B: 6 percent.

More preferably, the conditions of the second-stage separation by preparative high performance liquid chromatography are as follows: the sample injection amount is 1000 mu L; the chromatographic column is a C18 chromatographic column; mobile phase a was an aqueous solution containing 0.1% TFA, mobile phase B was an acetonitrile solution containing 0.1% TFA; the elution speed is 10.0 mL/min; the ultraviolet detection wavelength is double detection wavelength: 214nm and 280 nm; the elution conditions were: 0-40 min: mobile phase B: 5% -25%, 40-45 min: mobile phase B: 25% -90%, 40-45 min: mobile phase B: 25% -90%, 45-50 min: mobile phase B: 90%, 50-55 min, mobile phase B: 90% -5%, 55-65 min: mobile phase B: 5 percent.

Preferably, the conditions of the analytical high performance liquid separation in S3 are: the sample injection amount is 20 mu L; the chromatographic column is a C18 chromatographic column; mobile phase a was an aqueous solution containing 0.1% TFA, mobile phase B was an acetonitrile solution containing 0.1% TFA; the elution speed is 1.0 mL/min; the ultraviolet detection wavelength is double detection wavelength: 214nm and 280 nm; the elution conditions were: 0-40 min: mobile phase B: 5% -25%, 40-45 min: mobile phase B: 25% -90%, 45-50 min: mobile phase B: 90%, 50-55 min: mobile phase B: 5 percent.

The application of the calcium chelating peptide in preparing calcium supplement preparations or functional beverages is also within the protection scope of the invention.

Compared with the prior art, the invention has the following beneficial effects:

the calcium chelating peptide provided by the invention is safe and nontoxic, has better physicochemical activity compared with the traditional calcium supplement, has the calcium chelating capacity of 28.4mg/g, and can promote the calcium transport in a Caco-2 cell monolayer; can improve the absorption and bioavailability of calcium in human body, and can supplement collagen peptide as raw material for drug development and biological calcium supplement. The preparation method provided by the invention successfully obtains the calcium chelating peptide, can fully utilize fish product resources, is simple to operate, and provides a new way for high-value utilization of tilapia mossambica bones.

Drawings

FIG. 1 is a calcium chelating activity assay for each enzymatic time;

FIG. 2 is a separation chromatogram of a first separation and purification by preparative high performance liquid chromatography (peak C1 is 11-12.5 min, peak C2 is 12.5-16 min, peak C3 is 17-20 min, peak C4 is 21-22 min, peak C5 is 22-30 min, peak C6 is 20-38 min, peak C7 is 38-48 min, and peak C8 is 48-60 min);

FIG. 3 shows calcium sequestration activity for the first separation of peaks;

FIG. 4 is a separation chromatogram for a second separation preparation;

FIG. 5 shows chelating activity of 7 fractions from the second separation of C2 fraction;

FIG. 6 is a chromatogram of an analytical high performance liquid chromatography monomer preparation;

FIG. 7 is an amino acid sequence of calcium chelating peptide;

FIG. 8 is a graph of the effect of different concentrations of YQEPVIAPKL on cell viability;

FIG. 9 is a Caco-2 cell monolayer;

FIG. 10 is a graph of YQEPVIAPKL effect on calcium transport in Caco-2 cell monolayers, where a, b, c represent significant differences between groups at given time points (P < 0.05).

Detailed Description

The invention is further illustrated by the following examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples below, generally according to conditions conventional in the art or as suggested by the manufacturer; the raw materials, reagents and the like used are, unless otherwise specified, those commercially available from the conventional markets and the like. Any insubstantial changes and substitutions made by those skilled in the art based on the present invention are intended to be covered by the claims.