CN110669127A - Novel calcium chelating peptide and preparation method and application thereof - Google Patents
Novel calcium chelating peptide and preparation method and application thereof Download PDFInfo
- Publication number
- CN110669127A CN110669127A CN201910826228.9A CN201910826228A CN110669127A CN 110669127 A CN110669127 A CN 110669127A CN 201910826228 A CN201910826228 A CN 201910826228A CN 110669127 A CN110669127 A CN 110669127A
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- CN
- China
- Prior art keywords
- calcium
- enzymolysis
- calcium chelating
- peptide
- chelating peptide
- Prior art date
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Links
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- 229910052791 calcium Inorganic materials 0.000 title claims abstract description 119
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/52—Adding ingredients
- A23L2/66—Proteins
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/16—Inorganic salts, minerals or trace elements
- A23L33/165—Complexes or chelates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
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.
Description
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.
Example 1
This example provides a calcium chelating peptide having the amino acid sequence: YQEPVIAPKL ().
The calcium chelating peptide can be prepared by the following preparation method:
extraction of tilapia bone collagen
(1) Cleaning of tilapia mossambica framework raw material
Unfreezing the fishbone, removing impurities, cleaning with tap water, and removing the head of the fishbone.
(2) Fleshing treatment of tilapia mossambica skeleton
Cutting fishbone into 5cm, adding 5g neutral protease and 1000mL water into 1kg raw material, and performing enzymolysis for 2 hr (55 deg.C, pH 7.5) to remove broken meat attached to bone. After the enzymolysis is finished, impurities are removed by washing.
(3) Alkaline washing degreasing of tilapia mossambica bone
The fish bone is put into 0.1mol/LNaOH solution (w/v1:10), stirred for 24h and washed to be neutral.
(4) Decalcification treatment of tilapia mossambica bone
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.
(5) Acid treatment of tilapia mossambica bone
And (3) treating the decalcified fish bone with hydrochloric acid with the mass fraction of 4% for 18h, and washing the fish bone with water to be neutral to obtain the tilapia mossambica bone collagen.
Enzyme selection and time factor optimization of (II) bone collagen peptide
(1) Enzyme selection
Papain, trypsin, neutral protease and alkaline protease are respectively utilized to carry out enzymolysis for 3 hours under the respectively marked optimal pH, optimal temperature, enzyme bottom ratio of 1 percent and substrate concentration of 6 percent, and enzyme with the best enzymolysis effect and the highest tilapia bone collagen calcium chelating peptide activity is selected to carry out time factor optimization.
The results of tilapia mossambica bone collagen calcium chelating peptide activities of different enzymes are shown in table 1 below.
TABLE 1 determination of the calcium-chelating Activity of the enzymes
As can be seen from the analysis in Table 1, under the conditions that the concentration of a tilapia bone collagen substrate is 6%, the enzyme-substrate ratio of each enzyme is 1.0%, the enzymolysis pH is 7.0, and the enzymolysis temperature is 60 ℃, the chelating capacity of tilapia bone collagen calcium after papain enzymolysis is higher than that of neutral protease, alkaline protease and trypsin, and is 10.960mg/g, so papain is selected as a tool enzyme.
(2) Time factor optimization
Performing enzymolysis on the extracted fishbone collagen by using papain for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours and 6 hours respectively, wherein the enzymolysis conditions are that the pH value is 7.0, the temperature is 60 ℃, the ratio of enzyme to substrate is 1 percent and the concentration of substrate is 6 percent. Immediately carrying out water bath in boiling water for 10min to inactivate enzyme after enzymolysis, standing at room temperature for 30min to cool, then centrifuging in a centrifuge at 4000r/min for 20min, and freeze-drying the obtained supernatant to obtain tilapia mossambica bone collagen peptide freeze-dried powder under different time conditions.
After the calcium chelation activity is measured, the enzymolysis time with the optimal calcium chelation activity in the given conditions can be selected. The experimental results are shown in FIG. 1.
From the analysis of fig. 1, it can be seen that: under the conditions that the concentration of a tilapia mossambica bone collagen substrate is 6%, the papain base ratio is 1.0%, the enzymolysis pH is 7.0 and the enzymolysis temperature is 60 ℃, the activity of tilapia mossambica bone collagen calcium chelating peptide is gradually enhanced along with the extension of enzymolysis time, the trend of the enhancement of the activity of enzymolysis products chelated with calcium tends to be gentle when enzymolysis is carried out for 4 hours, the highest peak is reached when enzymolysis is carried out for 5 hours, and the calcium chelating activity slightly begins to be reduced when enzymolysis is carried out for 6 hours. This is probably due to the saturation of papain in the calcium chelation reaction, so that the calcium chelation activity of the enzymatic product is no longer enhanced.
Therefore, according to the experiment, the enzymatic hydrolysate obtained in 5h has the highest activity of chelating calcium, the calcium chelating concentration of the tilapia bone collagen calcium chelating peptide is 0.01 mu g/mL, and the calcium chelating capacity is 12.075 mg/g.
The results of the molecular weight distribution test for each enzymatic hydrolysis time are shown in Table 2.
TABLE 2 results of molecular weight at each digestion time
As can be seen from the analysis in Table 2, the molecular weight of the peptide fragment was mainly distributed in the range of 180-500Da regardless of the duration of the enzymatic hydrolysis. In the molecular weight ranges of 180-500Da and 500-1000Da, the proportion of molecules is gradually increased from 1h as the enzymolysis time is prolonged, the peak is reached at 4h, the molecular weight is slightly reduced at 5h, and the same molecular weight proportion as that at 5h is maintained at 6 h. This is probably due to the fact that the molecular weight ratio is not increased in the range of molecular weight 180-500Da and 500-1000Da because the enzymatic reaction is saturated. In addition, as the enzymolysis time is prolonged from 1h to 5h, the proportion of molecules with the molecular weight of more than 5000Da, the molecular weight range of 3000-5000Da and the molecular weight range of 2000-3000Da is gradually reduced, and the proportion is kept the same as that of 5h at 6h, because the peptide chain with larger molecular weight is gradually enzymolyzed and decomposed into the peptide chain with small molecular weight, and when the enzymolysis reaction reaches saturation, the proportion of larger molecular weight is not reduced. (III) separation and purification of tilapia bone calcium chelating peptide
(1) First separation of preparative high performance liquid chromatography
Freeze-drying the component with better calcium chelating ability obtained in the last step, adding first-stage water until the concentration is 1.24g/20mL, filtering with a 0.45-micrometer microfiltration water film, separating with high performance liquid phase, and measuring the calcium chelating activity of the separated substance to select a part with strong chelating activity. The preparation conditions of the high performance liquid chromatography are as follows:
the chromatogram results are shown in FIG. 2. And (3) carrying out first separation on the tilapia mossambica bone collagen calcium chelating peptide with the highest activity obtained after 5h enzymolysis in a preparative high performance liquid chromatography steel column to separate eight peak sections of C1, C2, C3, C4, C5, C6, C7 and C8. The peak section of C1 is 11-12.5 min, the peak section of C2 is 12.5-16 min, the peak section of C3 is 17-20 min, the peak section of C4 is 21-22 min, the peak section of C5 is 22-30 min, the peak section of C6 is 20-38 min, the peak section of C7 is 38-48 min, and the peak section of C8 is 48-60 min. The eight prepared components are collected, subjected to rotary evaporation and freeze drying (to obtain freeze-dried powder), and then subjected to calcium chelation activity determination by using an o-cresolphthalein colorimetric method, and the obtained determination result is shown in fig. 3.
Specifically, the specific process of calcium chelating activity determination by an o-cresolphthalein colorimetric method comprises the following steps:
1. tilapia bone collagen calcium chelating peptide treatment
Adding a calcium chloride solution and a phosphate buffer solution into a freeze-dried powder sample, wherein the final concentrations are respectively: the concentration of the sample to be tested is 500mg/L, 5mmol of calcium chloride solution and 20mmol of sodium phosphate buffer solution, and the pH value is 7.8. The mixture was incubated at 37 ℃ in a water bath for 120min with constant shaking. Centrifuging the mixed solution at 4000r/min for 20min, and taking the supernatant for later use. The sample group was not added as a blank control group.
2. And (3) detecting the activity of the tilapia mossambica bone collagen calcium chelating peptide by using an o-cresolphthalein colorimetric method.
2.1 preparation of o-cresolphthalein reagent by colorimetry
2.1.1 stock ethanolamine-borate buffer:
weighing 3.6g of boric acid into a 100mL volumetric flask, adding 10mL of distilled water and 10mL of ethanolamine, shaking until the boric acid is completely dissolved, and fixing the volume to 100mL by using the ethanolamine.
2.1.2 stock o-cresolphthalein solution:
80.0mg of o-cresolphthalein complexing agent is weighed into 100mL of a brown volumetric flask, 25mL of distilled water and 0.5mL of 1mol potassium hydroxide solution are added, and the mixture is shaken until the o-cresolphthalein complexing agent is completely dissolved. The volume was determined with 75mL of distilled water, 0.5mL of glacial acetic acid was added and shaken up.
2.1.3 stock 8-hydroxyquinoline solution:
5.0g of 8-hydroxyquinoline was weighed into a 100mL brown volumetric flask, dissolved in 95% ethanol and the volume was determined.
2.1.4 stock calcium standard (1000. mu.g/mL):
weighing about 2g of calcium carbonate (analytically pure) in a crucible, drying at 105 ℃ for 4h, taking out, putting into a dryer, cooling to room temperature, weighing 0.1g in a 100mL volumetric flask, adding 5mL of 0.5N HCl, diluting to constant volume with distilled water, and shaking up.
2.1.5 0.5N HCl：
43mL of concentrated HCl (analytical grade) was taken in a 1L volumetric flask, and the volume was determined with distilled water and shaken up.
2.1.6 Standard calcium working solution (100. mu.g/mL):
10mL of the stock calcium standard solution was aspirated into a 100mL volumetric flask and the volume was fixed with primary water.
2.1.7 working color developing solution:
6mL of the ethanolamine-borate buffer stock solution, 1.8mL of the 8-hydroxyquinoline stock solution and 6mL of the o-cresolphthalein solution are sucked into a 100mL volumetric flask, and the volume is fixed to 100mL by using primary water.
2.1.80.2 mol/L phosphate buffer solution preparation:
91.5mL of 0.2mol/L disodium hydrogen phosphate (17.907g of sample is dissolved to a constant volume of 250mL) and 8.5mL of 0.2mol/L sodium dihydrogen phosphate (0.78 g of sample is dissolved to a constant volume of 25mL) are mixed
2.2. Activity assay
In the experiment, the calcium activity is measured by using an o-cresolphthalein colorimetric method.
Respectively putting 0, 0.2, 0.4, 0.6, 0.8 and 1.0mL of standard calcium working solution (10 mu g/mL) into a 10mL test tube, respectively adding 1.0, 0.8, 0.6, 0.4, 0.2 and 0mL of first-grade water, then adding 5mL of working color development solution, shaking up, comparing color at 570nm within 0-15 min, and taking the obtained product as a standard line. When the sample is measured, 0.2-1.0 mL of sample solution is used according to the method, and when the sample is less than 1mL, 1mL is supplemented by first-grade water. The calculation formula of the bone collagen calcium chelating rate and the chelating capacity is as follows:
As can be analyzed from FIG. 3, C2 has high calcium chelating ability, which indicates that the activity of the chain segment calcium contained in the C2 component is high, the chain segment calcium is separated out at the peak-out time of 12.5-16 min, and the concentration of acetonitrile is about 28% -32%, so that a sample of the C2 peak segment is taken for freezing storage for further secondary separation and purification. According to the experimental result, the calcium concentration of the C2 peak segment is 0.022 mug/mL, and the calcium chelating capacity is 26.929 mg/g.
(2) Second separation in preparative high performance liquid chromatography
Freezing and storing the component with better calcium chelating ability obtained in the first separation by a preparative high performance liquid chromatography instrument, performing the second high performance liquid separation, and determining the calcium chelating activity of the collected substance to separate out the component with the strongest activity. The analysis conditions of the high performance liquid chromatography are as follows:
the chromatogram is shown in FIG. 4. As can be seen from the figure, the C2 component taken in the first preparation separation is subjected to the second separation on the preparative high performance liquid chromatography to separate seven peaks of C2-1, C2-2, C2-3, C2-4, C2-5, C2-6 and C2-7, and the peak emergence times of the seven peaks are 14-15 min, 16.5-17.2 min, 19.9-21 min, 22-23 min, 23.5-24.5 min, 25-27.5 min and 31.5-33 min respectively. The seven components obtained by the second liquid phase preparation are taken, and after rotary evaporation and freeze drying (to obtain freeze-dried powder), calcium activity is measured by using an o-cresolphthalein colorimetry, and the measurement result is shown in fig. 5.
As can be analyzed from FIG. 5, C2-7 has a high calcium chelating rate, which indicates that the chain segment in the C2-7 component has high calcium activity, the peak-off time is 31.5-33 min, and the acetonitrile concentration is about 20% when separated. The peak C2-7 has a calcium concentration of 0.036. mu.g/mL and a calcium chelating capacity of 43.018 mg/g. The C2-7 peak was picked and stored frozen for preparation of monomers on analytical high performance liquid chromatography equipment.
(3) Analytical high performance liquid phase separation and purification of monomer
And separating the optimal calcium chelating active component from the steel column, and preparing the monomer by utilizing analytical high performance liquid separation. Analytical high performance liquid phase analysis conditions were:
the chromatogram is shown in FIG. 6. As can be seen from the analysis of FIG. 6, a single monomer was prepared on analytical high performance liquid chromatography, collected and stored frozen for further activity determination and structural analysis. The peak-off time is 10.4-10.8 min, and the concentration of acetonitrile is about 20-20.5%.
(4) Comprehensive result analysis of tilapia mossambica bone collagen calcium chelating peptide activity
The data obtained from the experiments at each stage are shown in table 3.
TABLE 3 Tilapia bone collagen calcium chelating peptide Activity Synthesis results
From the analysis in table 3, it can be seen that through the four-stage experiment, the calcium chelating capacity of the tilapia bone collagen calcium chelating peptide is gradually enhanced after the selection of tool enzyme, the optimization of time factors and the separation and purification of two preparative high performance liquid chromatography, and the enhancing range of each time is larger and larger. This is due to the enhanced results of the enzymatic conditions and the isolation and purification of the active components.
(5) Tilapia bone collagen calcium chelating peptide sequence determination
The amino acid sequence of the tilapia mossambica bone collagen calcium chelating peptide is determined by an HPLC-MS/MS method, and the amino acid sequence is YQEPVIAPKL (Tyr-Gln-Glu-Pro-Val-Ile-Ala-Pro-Lys-Leu), as shown in figure 7.
Example 2
In this example, the calcium chelating ability of calcium chelating peptide and its effect on calcium transport in Caco-2 cell monolayer were studied, and the specific process is as follows.
(1) Peptide synthesis
Synthetic peptides (YQEPVIAPKL) were obtained from Synpeptide Co., Ltd (Nanjing, China) at a purity of more than 95% and their molecular weight was determined by LC/ESI-MS at 1157.627 Da.
Table 4 shows the proteins homologous to the calcium-chelating peptides.
TABLE 4 homologous proteins to calcium chelating peptides
The calcium chelating activity of the synthetic peptide was determined by using o-cresolphthalein colorimetry. YQEPVIAPKL at 28.4mg/g, indicating that the shorter peptide YQEPVIAPKL has more calcium chelating sites.
(2) Cell culture
Caco-2 cells were purchased from American Type Culture Collection (Rockville, Md., USA). Caco-2 cells of 40-44 generations were used in the experiment. Cells were cultured in complete medium (EMEM supplemented with 10% fetal bovine serum, 1% non-essential amino acids, 1% double antibody).
When the cells grew to 90% confluence, the Caco-2 cells were detached by treatment with trypsin-EDTA and plated at 3X 10 on 6-well transwell plates (3450, Corning Inc, USA) with polyester film (24 mm diameter)5cells/mL density seeding. The medium on the apical and basolateral side was changed every other day and cultured for 21 days. The integrity of the Caco-2 cell monolayer was confirmed by measuring the transepithelial electrical resistance (TEER) using an electrical resistance device (Millicell-ERS, Millipore Corp), and using a TEER higher than 300 Ω/cm2The monolayer of (a) was subjected to transport analysis.
(3) Toxicity test of cells
Cytotoxicity assays at various concentrations of YQEPVIAPKL were monitored by the MTT method. Caco-2 cells were cultured at 5X 104Individual cells/well density were plated in 96-well plates (Costar, Corning, NY) and plated in 5% CO2Incubate in the incubator at 37 ℃ for 24 hours. Subsequently, cells were treated with 100. mu.L of medium with different concentrations (25-1000. mu.g/mL) of peptide (YQEPVIAPKL) and incubated for 24 hours. After incubation, cells were killed by 100 μ L of 0.5mg/mL MTT solution for 2 hours. Then, 100. mu.L of dimethyl sulfoxide was used in place of the MTT solution. The 96-well plate was placed on a shaker for 15 minutes. Finally, the Light absorption was measured at a wavelength of 570nm using a spectrophotometer (EnspireXenon Light Module, Perkin-Elmer, Beacons field, U.K.). Sample concentrations with absorbance decreases by more than 10% compared to the control group are considered cytotoxic.
As shown in fig. 8, YQEPVIAPKL at the 6 tested concentrations were not toxic to cell viability.
(4) Study of calcium transport in Caco-2 cell monolayers
Differentiated Caco-2 cell monolayers were gently washed twice with pre-warmed HBSS and transferred to new 6-well plates containing 2mL HBSS, and 2mL HBSS was added on the top side. The Caco-2 cell monolayer was placed at 37 c,5％CO2was incubated for 30 minutes. Subsequently, 2mL of HBSS containing 150. mu.g/mL peptide (YQEPVIAPKL) and 150. mu.g/mL calcium was added to the apical side. (as shown in FIG. 9) while the control group was supplemented with only 2mL of HBSS containing 150. mu.g/mL. At different time intervals (30, 60, 120 and 180 minutes), 1mL of HBSS was collected from the outside of the substrate to determine the calcium content and immediately 1mL of HBSS was added to the outside of the substrate to keep the volume constant. Calcium content was measured by atomic absorption spectroscopy.
FIG. 10 shows the effect of YQEPVIAPKL on calcium transport in Caco-2 cell monolayers. Within 180 minutes, calcium absorption increased time-dependently. In the presence of YQEPVIAPKL, the calcium transport increased 202% at 30min compared to the control. As transit time increases.
The calcium chelating peptide 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.
Finally, it should be noted that the above embodiments are only representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above-described embodiments, and many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the present disclosure are to be considered within the scope of the claims of the present invention.
Sequence listing
<110> southern China university of agriculture
<120> novel calcium chelating peptide, preparation method and application thereof
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>10
<212>PRT
<213>2 Ambystoma laterale x Ambystoma jeffersonianum
<400>1
Tyr Gln Glu Pro Val Ile Ala Pro Lys Leu
1 5 10
Claims (10)
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.
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| CN102871121A (en) * | 2012-10-10 | 2013-01-16 | 中国食品发酵工业研究院 | Preparation method of oceanic ossein peptide calcium chelate biological calcium supplement |
| CN104774896A (en) * | 2015-04-15 | 2015-07-15 | 浙江海洋学院 | Preparation method for iron-chelated collagen peptide of hairtail fish-bones |
| CN106866785A (en) * | 2017-04-15 | 2017-06-20 | 福州大学 | A kind of calcium chelating peptide and preparation method thereof |
| CN109265536A (en) * | 2018-09-03 | 2019-01-25 | 华南农业大学 | A kind of calcium chelating peptide and its preparation method and application |
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| CN102871121A (en) * | 2012-10-10 | 2013-01-16 | 中国食品发酵工业研究院 | Preparation method of oceanic ossein peptide calcium chelate biological calcium supplement |
| CN104774896A (en) * | 2015-04-15 | 2015-07-15 | 浙江海洋学院 | Preparation method for iron-chelated collagen peptide of hairtail fish-bones |
| CN106866785A (en) * | 2017-04-15 | 2017-06-20 | 福州大学 | A kind of calcium chelating peptide and preparation method thereof |
| CN109265536A (en) * | 2018-09-03 | 2019-01-25 | 华南农业大学 | A kind of calcium chelating peptide and its preparation method and application |
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| CN111454347A (en) * | 2020-04-20 | 2020-07-28 | 广东海洋大学 | Peptide calcium chelate as well as preparation method and application thereof |
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