CN111454347A

CN111454347A - Peptide calcium chelate as well as preparation method and application thereof

Info

Publication number: CN111454347A
Application number: CN202010309244.3A
Authority: CN
Inventors: 吉宏武; 陈铭; 苏伟明; 刘书成; 郝记明
Original assignee: Guangdong Ocean University
Current assignee: Guangdong Ocean University
Priority date: 2020-04-20
Filing date: 2020-04-20
Publication date: 2020-07-28

Abstract

The invention discloses a preparation method of a peptide calcium chelate, which comprises the step of carrying out a chelation reaction on a protein source and a calcium source in water, wherein the protein source is Katsuwonus pelamis protein peptide, the parameter of the chelation reaction is set, the mass ratio of the protein source to the calcium source is 1: 3-3: 1, the chelation temperature is 30-70 ℃, the chelation time is 10-50 min, and the pH is 7-11.

Description

Peptide calcium chelate as well as preparation method and application thereof

Technical Field

The invention belongs to the technical field of biotechnology and aquatic product processing, relates to a deep processing technology of skipjack, and particularly relates to a peptide-calcium chelate and a preparation method and application thereof.

Background

Calcium is an indispensable macromineral for human body, has very important functions in all stages of juvenile, growth and old age of human, and has multiple physiological functions. Calcium deficiency is very common in people in underdeveloped areas, of which infants, children, pregnant women and the elderly are the main groups prone to calcium deficiency. Calcium deficiency presents different health problems at different stages of growth. When the calcium is continuously supplied insufficiently, the metabolism of a human body is disturbed, the normal life activities of the human body are influenced, and clinical symptoms can be caused in severe cases. Long-term calcium deficiency can cause the blood calcium concentration to be reduced, stimulate abnormal secretion of parathyroid cells, and excessive parathyroid hormone acts on osteoclasts to dissolve calcium in bones into blood so as to improve the blood calcium concentration, so that the bone calcium content is reduced, and finally, diseases such as osteoporosis, hyperosteogeny and the like are caused. In addition, insufficient calcium supply can affect endocrine, increase the incidence of cardiovascular and cerebrovascular diseases, hyperthyroidism and other diseases, and cause functional defects such as tooth dysplasia, slow blood coagulation, thyroid gland abnormality and the like.

Aiming at the current situation of calcium deficiency of people in underdeveloped areas, the research and development of calcium supplements are not completed until now, and a large number of calcium supplement products are researched and developed and applied to the health problem of calcium deficiency of human bodies. Calcium supplements are classified into 4 types, i.e., inorganic calcium, organic acid calcium, amino acid chelated calcium and peptide chelated calcium, according to their absorption rate in the body. However, most calcium supplement products only provide sufficient calcium content, but neglect the absorption mechanism of calcium, so that the calcium supplement products cannot be utilized efficiently. In order to improve the bioavailability of calcium supplement in human body, researchers have developed calcium amino acid and calcium peptide which are easier to be absorbed by human body, and the peptide has the characteristics of high absorption rate, low energy consumption, difficult carrier saturation and the like compared with free amino acid. Peptide chelated calcium is a monodentate, bidentate or polydentate chelate formed by peptide and calcium ions in different coordination modes, and is usually coordinated to form a five-membered ring or a six-membered ring structure. The peptide chelated calcium has the advantages of stable structure, strong polarity, good solubility, stable heating, high bioavailability and the like. Compared with the 3 calcium supplements, the peptide chelated calcium has the highest bioavailability, is generally directly absorbed by intestinal tracts in an integral molecular form, then enters blood circulation from portal veins, and slowly releases calcium ions according to the needs of organisms. The absorption mode of the peptide chelated calcium is not easy to saturate, the absorption speed is high, the energy consumption is low, and the biological potency of the calcium in the peptide chelated calcium can be effectively improved. The peptide chelated calcium is the most potential calcium supplement product at present, but most of the peptide chelated calcium is still in the research stage, so that the peptide chelated calcium has larger development space and market potential.

Auxis thazard (Auxis thazard) is a common green tangerine peel red meat fish in China, belonging to Auxis thazard of tuna family. The flat-rudder bonito is a low-value tuna, cannot be favored by consumers due to relatively sour meat quality, high dark meat proportion, heavy fishy smell and poor taste, is sold in a fresh food form in the market, and is mostly used as a production raw material of canned tunas. But the nutritional value of the tuna is not much different from that of the tuna, the tuna has high protein, low fat, rich omega-3 fatty acid, vitamins and minerals, large fishing amount and low price, so the tuna has great development potential. In addition, the bonito hydrolyzate has reasonable amino acid composition, contains essential amino acid content of 40% or more of the total amino acid content, and contains amino acids having strong metal chelating ability such as aspartic acid, glutamic acid, histidine, proline, etc. Therefore, the hydrolysis product of the bonito is a high-quality protein source which has certain metal chelating capacity and is to be developed.

Disclosure of Invention

The extensive research and development of different types of calcium supplement products can relieve the health problems caused by calcium deficiency to a certain extent. However, in the current calcium supplement products on the market, the types of protein sources of peptide chelated calcium are limited, the products are single, and compared with other types of calcium supplement products, the products are expensive and low in known degree, so that the development of the products is limited. Aiming at the development trend of calcium supplement products and the practical problem of low development and utilization degree of the bonito protein, the invention provides a peptide-calcium chelate, a preparation method and application thereof, and explores the high-valued utilization of the bonito protein source and the development of novel peptide-calcium products.

Firstly, the invention provides a preparation method of the peptide calcium chelate, which comprises the step of carrying out a chelating reaction on a protein source and a calcium source in water, wherein the protein source is selected from an Auxis thazard protein peptide; setting the parameters of the chelation reaction: the mass ratio of the protein source to the calcium source is 1: 3-3: 1, the chelation temperature is 30-70 ℃, the chelation time is 10-50 min, and the pH is 7-11.

As a further preferable method for producing the peptide-calcium chelate, the bonito protein peptide is produced by subjecting bonito to enzymatic hydrolysis with a protease; the protease is selected from papain and flavourzyme; and (3) carrying out enzyme deactivation on the enzymolysis product, then centrifuging, deoiling and decoloring the supernatant, filtering by using a ceramic membrane and an ultrafiltration membrane, and concentrating and drying to obtain the bonito protein peptide with the molecular weight not higher than 5000U.

As a further preferable method for producing the peptide calcium chelate, a preferable method for producing the bonito protein peptide is given:

taking meat of skipjack, mincing, homogenizing, and performing enzymolysis with papain and flavourzyme; the enzymolysis conditions are as follows: the temperature is 50 ℃, the adding amount of papain is 500U/g, the adding amount of flavourzyme is 200U/g, the pH is 6.5, and the enzymolysis time is 3 hours;

carrying out water bath enzyme deactivation on the enzymolysis product, then centrifuging for 15min at the centrifugation temperature of 4 ℃, centrifuging and deoiling the obtained supernatant, and decoloring perlite, wherein the addition amount of the perlite is 3%, then filtering by a ceramic membrane and an ultrafiltration membrane, the aperture of the ceramic membrane is 0.2 mu m, and the molecular weight cut-off of the ultrafiltration membrane is 5000U;

concentrating and spray drying to obtain the bonito protein peptide, wherein the content of the protein peptide with the molecular weight of 180-5000U is not less than 70%, and the content of oligopeptide with the molecular weight of less than 1000U is not less than 60%.

As a further preference of the preparation method of the peptide calcium chelate, the parameters of the chelation reaction are set as follows: the mass ratio of the protein source to the calcium source is 2:1, the chelation temperature is 50 ℃, the chelation time is 20min, and the pH value is 9.

And (3) separating and purifying protein peptides with high calcium binding activity from the Katsuwonus pelamis protein peptides by using Sephadex G-15 gel chromatography and RP-HP L C, namely calcium binding active peptides Glu-Pro-Ala-His and Tyr-Asp-Thr contained in the Katsuwonus pelamis protein peptides.

The invention further discloses a mode of combining the Glu-Pro-Ala-His with calcium ions, namely, the carboxylic acid group of the Glu is combined with the calcium ions in a double-tooth mode, and the carboxylic acid group and the amino group of the His are chelated with the calcium ions in an α mode to form a five-ring structure.

Furthermore, the invention provides a peptide calcium chelate, which is prepared by the preparation method of the peptide calcium chelate.

Also, the present invention claims a protein peptide derived from bonito oblast (Auxis thazard) containing Glu-Pro-Ala-His and Tyr-Asp-Thr, calcium-binding active peptides;

the Glu-Pro-Ala-His is combined with calcium ions in a mode that carboxylic acid groups of Glu are combined with calcium ions in a double-tooth mode, and carboxylic acid groups and amino groups of His are chelated with calcium ions in an α mode to form a five-ring structure.

Also, the present invention claims the use of a protein peptide, which is an Auxis thazard (Auxis thazard) protein peptide, for preparing a peptide calcium chelate.

Furthermore, the invention claims the use of a peptide calcium chelate or a medicament containing the peptide calcium chelate for promoting the growth and development of bones.

The invention has the following beneficial effects or advantages:

the method takes skipjack (Auxis thazard) as a raw material, prepares protein peptide by an enzymatic hydrolysis method, optimizes the preparation process of the skipjack protein peptide chelated calcium, then separates and purifies the protein peptide to obtain calcium binding peptide, analyzes the binding characteristic of the peptide and calcium ions, finally establishes a low-calcium animal model, and inspects the influence of the skipjack protein peptide chelated calcium on the bone growth of a rat.

(1) Compositional property analysis and nutritional evaluation show that the bonito protein peptide is a high-quality nutritional base material with high protein, low fat, comprehensive amino acid composition and high essential amino acid score, has high peptide content, particularly high small peptide content, and 67.99% of oligopeptide with the molecular weight less than 1000U, and is a high-quality calcium source for preparing peptide chelated calcium.

(2) Through research on reaction conditions such as chelating time, chelating temperature, pH and peptide-calcium mass ratio, the preparation process conditions of the bonito protein peptide chelated calcium are as follows: the chelating temperature is 50 ℃, the chelating time is 20min, the pH is 9, the mass ratio of the peptide calcium is 2:1, under the condition, the chelate yield reaches 41.06%, and the chelate calcium content is 14.98%. Various characterization results prove that the bonito protein peptide and calcium ions have chelation reaction to form a peptide chelated calcium product.

(3) Two short peptides having high calcium-binding activity were isolated and purified from bonito protein peptide by Sephadex G-15 gel chromatography and RP-HP L C, and the amino acid sequences thereof were identified by Q-TOF as Glu-Pro-Ala-His (MW: 453.3U) and Tyr-Asp-Thr (MW: 398.1U), respectively, wherein Glu-Pro-Ala-His is a novel calcium-binding active peptide which binds to calcium ions in such a manner that the carboxylic acid group of Glu binds to calcium ions in a bidentate pattern and the carboxylic acid group and amino group of His bind to calcium ions in a α pattern, and based on this result, a hypothetical molecular model of peptide-calcium chelate was constructed.

(4) Animal experiments show that the bonito protein peptide chelated calcium can improve the malnutrition of rats, increase the weight of the rats, effectively inhibit the A L P activity, and simultaneously improve the absorption rate of calcium in the rats.

Drawings

FIG. 1 is a graph showing the temperature effect of chelation reaction of a bonito protein peptide with calcium ions.

FIG. 2 is a graph showing the influence of time on chelation reaction of Katsuwonus Pelamis protein peptide with calcium ion.

FIG. 3 is a graph showing the influence of the peptide-calcium mass ratio of the Patinophans protein peptide on the chelation reaction of calcium ions.

FIG. 4 is a graph showing the influence of pH on chelation reaction of Katsuwonus Pelamis protein peptide with calcium ion.

FIG. 5 is a scanning curve of a skipjack protein peptide and a peptide calcium chelate in a wavelength range of 190-400 nm.

FIG. 6 is a graph showing the change of the mass of bonito protein peptide and peptide-chelated calcium with temperature by TG thermogravimetric analysis.

FIG. 7 is an X-ray diffraction pattern of Katsuwonus Pelamis protein peptide and calcium peptide chelate.

FIG. 8 is a scanning electron micrograph of Katsuwonus Pelamis protein peptide.

FIG. 9 is a scanning electron micrograph of Katsuwonus Pelamis protein peptide chelated calcium.

FIG. 10 is a graph showing the spectral analysis of Katsuwonus Pelamis protein peptide.

Fig. 11 is a graph showing the spectrum analysis of the bonito protein peptide chelated calcium.

FIG. 12 is a Sephadex G-15 gel separation chromatogram of Aureobasidium album oligopeptide.

FIG. 13 is a graph showing the calcium binding activity of fractions F1, F2, F3, F4 and F5 obtained by gel separation of Aureopsis ocellata oligopeptide Sephadex G-15.

FIG. 14 is a primary mass spectrum of Katsuwonus Pelamis oligopeptide fraction F42.

FIG. 15 is an amino acid sequence identification spectrum of Katsuwonus Pelamis oligopeptide fraction F42.

FIG. 16 is a primary mass spectrum of Katsuwonus Pelamis oligopeptide fraction F43.

FIG. 17 is an amino acid sequence identification spectrum of Katsuwonus Pelamis oligopeptide fraction F43.

FIG. 18 shows that the chelate of the protein peptide and peptide calcium of Katsuwonus Pelamis is 400-4000 cm^-1Infrared spectrum over a range of wavelengths.

FIG. 19 is a first-order mass spectrum analysis of Katsuwonus Pelamis protein peptide chelated calcium.

FIG. 20 is a second-order mass spectrometry of bonito protein peptide chelated calcium.

Fig. 21 is a schematic view of a molecular mechanism of bonito protein peptide chelated calcium.

FIG. 22 shows the effect of bonito protein peptide chelated calcium on the apparent absorption rate of calcium in rats.

FIG. 23 is a three-dimensional view of the distal femur of rats in the low dose group (a: cortical and cancellous bone mass, b: trabecular longitudinal bone mass, c: trabecular circumferential bone mass).

FIG. 24 is a three-dimensional view of the distal femur of a blank group of rats (a: cortical and cancellous bone mass, b: trabecular longitudinal bone mass, c: trabecular circumferential bone mass).

FIG. 25 is a three-dimensional plot of the distal femur of rats in the medium dose group (a: cortical and cancellous bone mass, b: trabecular longitudinal bone mass, c: trabecular circumferential bone mass).

FIG. 26 is a three-dimensional plot of the distal femur of rats in the peptide + calcium group (a: cortical and cancellous bone mass, b: trabecular longitudinal bone mass, c: trabecular circumferential bone mass).

FIG. 27 is a three-dimensional view of the distal femur of rats in the high dose group (a: cortical and cancellous bone mass, b: trabecular longitudinal bone mass, c: trabecular circumferential bone mass).

FIG. 28 is a three-dimensional plot of the distal femur of rats in the calcium carbonate group (a: cortical and cancellous bone mass, b: trabecular longitudinal bone mass, c: trabecular circumferential bone mass).

Detailed Description

In order to facilitate understanding of the objects, technical solutions and effects of the present invention, the embodiments of the technical solutions of the present invention will be further described in detail with reference to the examples.

Example one, preparation and characterization of skipjack protein peptide powder

1. Preparation method of skipjack protein peptide powder

This example shows a preferred method for preparing bonito (Auxis thazard) with the following operation. Taking meat of skipjack, mincing, homogenizing, and then carrying out enzymolysis by papain and flavourzyme under the conditions as follows: the temperature is 50 ℃, the adding amount of papain is 500U/g, the adding amount of flavourzyme is 200U/g, the pH value is 6.5, and the enzymolysis time is 3 hours. And (3) carrying out water bath enzyme deactivation on the enzymolysis product, then centrifuging for 15min (3500g and 4 ℃), deoiling the obtained supernatant by a tube centrifuge, decoloring perlite (the addition amount is 3%), then filtering by a ceramic membrane (the aperture is 0.2 mu m) and an ultrafiltration membrane (5000U), and finally concentrating and spray drying to obtain the bonito oligopeptide powder.

2. Protein and amino acid analysis of skipjack protein peptide powder

(1) Protein content determination

According to GB 5009.5-2016 (determination of protein in food), the protein content of the Katsuwonus Pelamis protein powder is up to 88.91 percent, the protein content is higher than that of silver carp protein powder, shrimp protein powder and soybean protein powder, and the fat content is only 0.13 percent, which shows that the Katsuwonus Pelamis protein powder is a high-quality nutritional base material with high protein and low fat. The protein peptide powder of the skipjack prepared by enzymolysis is high-quality protein powder, the peptide content is as high as 71.96 percent, and the protein peptide powder is a good peptide source for preparing peptide chelated calcium.

(2) Analysis of amino acid composition

The amino acid composition of the bonito protein peptide powder was analyzed with reference to GB-5009.124-2016, "determination of amino acids in foods". The results are shown in Table 1. The content of 5 amino acids with calcium binding activity, including aspartic acid, glutamic acid, lysine, arginine and histidine, in the Katsuwonus Pelamis protein peptide is 47.73%, and accounts for about half of the total content of the amino acids. Therefore, the bonito protein peptide powder should have a strong calcium chelating ability. In addition, 16 amino acids including methionine, valine, lysine, isoleucine, phenylalanine, leucine and threonine, 7 essential amino acids and arginine which is a conditionally essential amino acid are detected from the skipjack protein peptide powder. The total content of amino acids is 94.81g/100g, and the content of essential amino acids is 37.64g/100g, which shows that the peptide powder has comprehensive nutrition and high purity. Wherein the content of lysine in the Katsuwonus Pelamis protein peptide is up to 10.61g/100 g. The ratio (WEAA/WTAA) of the amount of amino acids necessary for human body in the skipjack protein peptide powder to the total amount of amino acids is not much different from the reported ratio, and is about 40 percent and higher than the standard (35.38 percent) of FAO/WHO; the ratio of essential amino acid to non-essential amino acid (WEAA/WNEAA) is 65.83%, and meets the FAO/WHO recommended protein nutrition evaluation standard.

TABLE 1 amino acid composition analysis of skipjack protein peptide

(3) Molecular weight distribution of protein

The molecular weight distribution of the skipjack Protein peptide is measured on a Protein purification system by using a Protein-PakTM 60A (7.8 × 300mm) Protein analysis column under the elution condition that a mobile phase is deionized water, an ultraviolet detection wavelength is 220nm and an elution speed is 1m L/min, standard substances are cytochrome C (12384U), bovine insulin (5733U), growth inhibin 28(3148U), growth inhibin 14(1637U), hippuryl-histidyl-leucine (429) and ethanyl-ethionine (189), standard substances with different molecular weights are eluted from the analysis column, a molecular weight determination standard curve is determined according to the corresponding relation between the elution volume of the standard substances and L og MW of the standard substances, a molecular weight determination standard curve is obtained according to the corresponding relation between the elution volume of the standard proteins and L og MW, a molecular weight determination standard curve is calculated according to the molecular weight determination standard curve, the molecular weight distribution of the skipjack Protein peptide is calculated, the molecular weight distribution of the skipjack Protein peptide is in a high-quality skipjack powder, the molecular weight distribution is 67.99%, and the molecular weight of the high-quality skipjack Protein is less than 1000.

TABLE 2 molecular weight distribution of skipjack protein peptide

In conclusion, the skipjack protein peptide is a high-nutritional-value base material with high protein content, low fat content, high peptide content and a molecular weight mainly within 5000U. The amino acid composition of the bonito protein peptide is comprehensive, and the content is uniform. The peptide content of the Katsuwonus Pelamis protein peptide, particularly the content of small peptide is very high, the oligopeptide with the molecular weight of less than 1000U accounts for 67.99 percent, and the Katsuwonus Pelamis protein peptide is a high-quality protein source for preparing peptide chelated calcium.

Example two, preparation of skipjack protein peptide chelated calcium and structural characterization thereof

The preparation yield of the protein peptide chelated calcium is closely related to the amino acid composition and the molecular weight of the peptide source, the type of the calcium source and the preparation conditions. The bonito protein peptide chelate calcium prepared in this example was selected from the bonito protein peptide of example 1 as a protein source, and the calcium source was of various types, among which calcium chloride and calcium hydroxide were most widely used, and calcium chloride was selected as the calcium source in this example. In this example, the kind of the calcium source is not particularly limited, and those skilled in the art can prepare the bound calx by selecting one or more specific calcium sources based on the technical idea of the present invention.

1. Preparation of peptide calcium chelate

Weighing 1.0g of skipjack protein powder and 2.0g of calcium chloride, fully dissolving in 50m L deionized water, adjusting pH, placing in a constant-temperature water bath shaker for chelation reaction, centrifuging after the reaction is finished to remove calcium hydroxide precipitate (4000r/min, 10min), performing rotary evaporation and concentration on supernatant, adding absolute ethyl alcohol until the volume is more than 90%, performing alcohol precipitation and centrifuging (8000r/min, 25min), and finally freeze-drying the obtained precipitate to obtain a chelate finished product (-50 ℃, 48 h).

The influence of the chelating temperature (30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃), the chelating time (10min, 20min, 30min, 40min, 50min), the pH (7, 8, 9, 10, 11) and the mass ratio of peptide to calcium (1:3, 1:2, 1:1, 2:1, 3:1) on the chelating reaction are respectively examined by taking the chelate yield and the chelate calcium content as indexes.

The temperature effect graph of the chelation reaction of the bonito protein peptide and calcium ions is shown in fig. 1. As can be seen from FIG. 1, with the increase of the chelating temperature, the chelate yield increases first and then decreases, and the change trend of the chelate calcium content and the chelate yield is consistent. The peptide calcium chelation reaction is an endothermic reaction, and in the temperature rise process of 30-50 ℃, the chelate yield and the chelate calcium content gradually increase and reach peak values respectively of 38.55% and 13.17% when the chelation temperature is 50 ℃. When the chelating temperature exceeds 50 ℃, the chelate yield and the chelate calcium content are reduced to different degrees, probably because the overhigh temperature is not beneficial to the forward reaction of peptide calcium chelating, and the amino acid or small peptide can generate the ammonia-carbonyl reaction to compete with calcium ions. Therefore, 50 ℃ is the optimum temperature for the chelation reaction of the bonito protein peptide and calcium ions.

The time effect graph of the chelation reaction of the bonito protein peptide with calcium ions is shown in fig. 2. As can be seen from FIG. 2, with the increase of the chelating time, the chelate yield and the chelate calcium content increase first and then decrease, and at 20min, both of them reach the highest values. The reason may be that the chelation time is too short and the chelation reaction is insufficient; the chelate time is too long and the formed chelate may be decomposed. Therefore, 20min was chosen as the optimal chelation time. The chelate yield and the chelate calcium content have gentle change trends along with the extension of the chelation time, which shows that the calcium chelation reaction is rapid, and the chelation time has little influence on the chelate rate.

The influence curve of the mass ratio of peptide to calcium in the chelation reaction of the bonito protein peptide and calcium ions is shown in fig. 3. As can be seen from FIG. 3, with the increase of the mass ratio of peptide to calcium, the chelate yield and the chelate calcium content both tend to be flat after increasing, and when the mass ratio of peptide to calcium is 2:1, the chelate yield reaches the highest value (41.06%), and the chelate calcium content (14.98%) also approaches the highest value. Under the condition of certain calcium chloride addition mass, the increase of the addition mass of the bonito protein peptide provides sufficient calcium binding sites for chelation reaction, and the bonito protein peptide is saturated when the mass ratio of the calcium to the peptide is 2: 1. Therefore, the optimal condition is selected as the mass ratio of the peptide to the calcium is 2:1 by comprehensively considering the utilization rate of the protein peptide and the calcium chloride.

The pH effect of the chelation reaction of the bonito protein peptide with calcium ions is shown in FIG. 4. As can be seen from FIG. 4, pH is one of the main factors affecting the peptide calcium chelation reaction. Within the range of pH 7-9, the chelate yield and the chelate calcium content gradually increase with the increase of pH value, probably because the hydrogen ion concentration decreases with the increase of pH, the capability of competing with free calcium ions in the solution for supplying power groups is weakened, and the coordination capability of the calcium ions with-NH 2 and-COOH is enhanced. At pH 9, chelate yield and chelate calcium content both reached a maximum. Under strong alkaline conditions, OH-and electron-donating groups compete for calcium ions to form calcium hydroxide precipitates, and chelate formation is not facilitated. Therefore, the pH of 9 was selected as the optimum pH for the bonito protein peptide chelated calcium.

2. Structural characterization of peptide calcium chelate

In order to confirm the formation of a product prepared from the bonito protein peptide chelated calcium and provide a research basis for the structure-activity relationship between the peptide and the calcium, the structure of the peptide chelated calcium is characterized by adopting an ultraviolet spectrum, TG thermogravimetry, X-ray diffraction and scanning electron microscope-energy spectrum analysis after the peptide calcium chelate is prepared.

The scanning curve of the skipjack protein peptide and the peptide calcium chelate in the wavelength range of 190-400 nm is shown in fig. 5. The ultraviolet absorption spectrograms of the protein peptide and the peptide calcium chelate are obviously different. The protein peptide has obvious absorption peaks at 190-220 nm and 250-280 nm, and after the protein peptide is combined with calcium ions, the maximum ultraviolet absorption peak at 195nm is blue-shifted to 190nm, so that the change is caused, probably because Ca is generated after the peptide and the calcium ions are subjected to chelation reaction²⁺Form a coordinate bond with N, O in the peptide, affecting the carbonyl (C ═ O) n → pi electron transition on the peptide bond, thereby causing it to undergo a blue shift; in addition, the ultraviolet absorption peak position and the intensity of the protein peptide at 250-280 nm are basically unchanged, which shows that the reaction does not influence the pi → pi electron transition of conjugated double bonds,it is shown that tryptophan and tyrosine are not involved in the chelation reaction of protein peptide and calcium ion.

The results of thermogravimetric analysis of the change curve of the mass of bonito protein peptide and peptide-chelated calcium with temperature by TG are shown in fig. 6. As the temperature increases, the mass of the samples gradually decreases, and as can be seen from their weight change, the thermal decomposition is mainly divided into two steps: before 105 ℃, the water loss is mainly caused, the change trends of the oligopeptide and the peptide calcium are basically consistent, and the water content is about 4%; the organic components start to be gradually decomposed within the temperature range of 200-480 ℃, and the decomposition rate of the protein peptide is obviously higher than that of the peptide calcium chelate in the process, so that the peptide calcium chelate has stronger thermal stability relative to the protein peptide. In addition, the ash content of the peptide calcium chelate is obviously higher than that of the protein peptide, and the increased part is mainly calcium element, which indicates that calcium ions are combined with the protein peptide.

An X-ray diffraction pattern of the bonito protein peptide and the peptide calcium chelate is shown in fig. 7, and compared with the protein peptide, the peptide calcium chelate has a higher diffraction peak and higher intensity, and a new sharp diffraction peak appears. The result shows that the protein peptide of the skipjack is chelated with calcium and generates a characteristic crystal structure containing calcium ions, and the crystal form is good.

The microscopic surface structures (2K ×) of the peptide powder and the peptide calcium chelate powder were observed by a scanning electron microscope, and it can be seen from FIGS. 8 and 9 that, after the oligopeptide of bonito, which is a flat rudder, chelates calcium ions, the oligopeptide was changed from a smooth porous structure into a dense granular material, which may be caused by the coordination and bonding of the peptide to the calcium ions and the entrapment of calcium by the porous space of the peptide.

The spectrum analysis of the bonito protein peptide and the peptide chelated calcium is shown in fig. 10 and 11. The energy spectrum analysis result shows that the peptide contains C, N, O elements and the like, and basically does not contain Ca element; the existence of Ca element in the peptide calcium chelate is proved by that the peptide calcium chelate contains a large amount of Ca element besides C, N, O element. Quantitative analysis of the elements shows that (Table 3), the normalized mass percentage of calcium elements after the peptide chelates calcium ions is increased from 0.37 +/-0.11% to 15.08 +/-0.82%. In conclusion, the occurrence of the chelation reaction was further confirmed by scanning electron microscopy and energy spectrum analysis.

Table 3. pulverata bonito protein peptide and peptide chelated calcium energy spectrum analysis results

In this example, through research on a preparation process of bonito protein peptide chelated calcium, it is determined that the preparation conditions are as follows: the chelating temperature is 50 ℃, the chelating time is 20min, the pH is 9.0, the mass ratio of the peptide calcium is 2:1, under the condition, the chelate yield reaches 41.06%, and the chelate calcium content is 14.98%. Various characterization results prove that the bonito protein peptide and calcium ions have chelation reaction to form a peptide chelated calcium product.

EXAMPLE III isolation and purification of calcium binding peptides

In this example, a protein peptide having a high calcium binding activity was isolated and purified from a bonito protein peptide by Sephadex G-15 gel chromatography and RP-HP L C, and its binding property to calcium ions was investigated by infrared spectroscopy and mass spectrometry.

The bonito oligopeptides were separated by Sephadex G-15 gel (FIG. 12) to give 5 different fractions, designated F1, F2, F3, F4, and F5, and their calcium binding activities are shown in FIG. 13. As the calcium binding capacity of the peptide gradually increased with decreasing molecular weight, the first 4 fractions had stronger calcium binding activity, of which fraction F4 showed the strongest calcium binding capacity (73.6. + -. 4.6mg/g), and fraction F5 had significantly lower calcium binding activity than the other 4 fractions, presumably the main components of this fraction being possibly amino acids and ionic impurities. The calcium binding activity of the peptides isolated by gel chromatography in this example is inversely related to the molecular weight. Fraction F4 was subsequently collected for further isolation.

The fraction F4 obtained by Sephadex G-15 gel separation is further separated by semi-preparative RP-HP L C to obtain four peaks F41, F42, F43 and F44, wherein the fractions F42 and F43 both show stronger calcium binding activity, namely 76.8 +/-4.5 mg/G and 74.2 +/-3.9 mg/G respectively, and the calcium binding activity is higher than that of protein peptide (0.134mmol/G) derived from Antarctic krill and is slightly lower than that of whey protein hydrolysate.

The first-order mass spectrum result of the component F42 is shown in FIG. 14, and the peaks with mass-to-charge ratios (m/z) of 453.3137 and 227.1594 respectively correspond to parent ions [ m + H ] of the component F42]⁺And [ m +2H]²⁺Then selecting the most abundant parent ion [ m + H ]]⁺(m/z 453.3137) secondary mass spectrometry was performed. The primary mass spectrum of fraction F43 is shown in FIG. 16, and the signal peak with the largest mass-to-charge ratio (m/z 398.0709) is selected as the parent ion [ m + H ]]⁺Secondary mass spectrometry was performed.

In tandem mass spectrometry, the amino acid sequence is mainly predicted by fragment ions in secondary mass spectrometry, and the generally formed fragment ions are mainly divided into two types: x, y and z represent fragmentation at the C-terminus, and a, b and C represent fragmentation at the N-terminus. In addition, since amide bonds in polypeptide chains are more easily cleaved, there is a greater chance of b-type and y-type ions occurring. According to this rule, the parent ion [ M + H ] can be estimated]⁺(M/z-453.3137) and parent ion [ M + H]⁺(m/z-398.0709). As can be seen from fig. 15, the fragment ion b2 had an m/z of 227.1599 corresponding to the relative molecular weight of fragment Glu-Pro-, and the fragment ion y2 had an m/z of 225.2504 corresponding to the relative molecular weight of fragment-Ala-His. Similarly, fragment ion b3(m/z 334.4581) corresponds to the relative molecular weight of fragment Glu-Pro-Ala-, fragment ion y1(m/z 154.1621) corresponds to the relative molecular weight of fragment-His, and fragment ion y3(m/z 322.2369) corresponds to the relative molecular weight of fragment-Pro-Ala-His. The analysis result of the mass spectrum shows that the peptide is tetrapeptide consisting of glutamic acid, proline, alanine and histidine, and fragment ions are analyzedb2, b3, y1, y2 and y3, and the amino acid sequence thereof was identified as glutamic acid-proline-alanine-histidine (Glu-Pro-Ala-His, MW: 453.3U). By analogy, by analysing parent ions [ M + H ]]⁺(m/z-398.0709) fragment ions b1, b2 and y1, y2 (fig. 17), the peptide was determined to be a tripeptide consisting of tyrosine, aspartic acid and threonine, and the amino acid sequence thereof was identified as tyrosine-aspartic acid-threonine (Tyr-Asp-Thr, MW: 398.1U). In this example, glutamic acid-proline-alanine-histidine (Glu-Pro-Ala-His), which is a hitherto unreported tetrapeptide, was selected and its binding properties to calcium ions were investigated by infrared spectroscopy and mass spectrometry.

The concentration of the complex of the skipjack protein peptide and the peptide calcium is 400-4000 cm^-1An infrared spectrogram in a wavelength range is shown in 18, and compared with tetrapeptide (Glu-Pro-Ala-His), the infrared spectrogram after the peptide is chelated with calcium is obviously shifted, which shows that after the peptide (Glu-Pro-Ala-His) and the calcium are subjected to chelation reaction, certain groups of amino acid participate in the reaction, and the vibration frequency of the amino acid is changed to cause the change of an absorption peak. In FIG. 18, characteristic bands of the peptide including the amide I band and the amide II band are shown, which are located at 1638cm^-1、1576cm^-1To (3). Characteristic region 3298cm^-1Near the N-H stretching vibration, in the infrared spectrogram of peptide (Glu-Pro-Ala-His) chelated calcium, the wave band is shifted to 3373cm^-1(due to the stretching vibration of N-H), it was shown that during chelation, blue shift occurred and the peak became broad, which is the characteristic frequency of ammonium salt, probably due to the substitution of Ca atom for H atom. The amide I band is mainly caused by C ═ O stretching vibration, and the absorption peak of C ═ O in the spectrogram is 1638cm^-1Move to 1670cm^-1And the strength is weakened, which indicates that after the peptide is chelated with calcium, the stretching vibration of C ═ O can be inhibited, and certain chemical action exists between C ═ O and calcium ions, and the action can be generated by coordination chelation of carboxylic acid and calcium ions. The amide II band is caused by in-plane deformation vibration of N-H, and the absorption peak of N-H in the infrared spectrogram of the peptide is 1576cm^-1Move to 1593cm^-1Thus N-H provides coordination sites for calcium ions. In addition, the absorption peak of C-N is from 1408cm^-1Move to 1420cm^-1The absorption peak of C-O is 1055cm^-1Move to 1092cm^-1This may be due to the fact that the calcium ion is acted upon by the common electron pair of the nitrogen atom to enhance the dipole property of the C-N bond, indicating that C-N, C-O is also involved in the reaction.

The peptide-calcium chelates were analyzed by primary and secondary mass spectrometry using Xevo G2-XS QTof tandem quadrupole time-of-flight mass spectrometry, the results of which are shown in fig. 19 and 20. In FIG. 19, the parent ion [ M + Ca ]]²⁺、[M+2Ca-3H]²⁺And [ M +2Ca-3H ]]⁺Corresponding to signal peaks of M/z 246.1963, M/z 264.7375 and M/z 496.4689, respectively, and at the same time, a parent ion [ M + H ] of peptide (Glu-Pro-Ala-His) appeared]⁺(m/z 453.3146), consistent with the results of fig. 14, indicating that the peptide may bind to one calcium or two calcium and that the difference in the binding pattern results in parent ions of different mass to charge ratios. Then selecting parent ion [ M +2Ca-3H ]]⁺(m/z 496.4689) secondary mass spectrometry was performed. Parent ion [ M +2Ca-3H]⁺Smaller fragments were generated by electrospray ionization fragmentation, and the results are shown in fig. 20, wherein the mass spectrum peaks m/z-192.2381, m/z-263.3283, and m/z-360.4581 correspond to the ion fragments [ y1+ Ca-2H ] respectively]⁺、[y2+Ca-2H]⁺、[y3+Ca-2H]⁺This indicates that the binding site for calcium ion can be located in the C-terminal fragment of-His, -Ala-His or-Pro-Ala-His, with the presence of the b-type fragment ion [ b2+ Ca-NH3 ]]⁺(m/z-266.1963), which identifies one of the calcium ion binding sites on the N-terminal fragment of Glu-Pro-. From [ y1+ Ca-2H]⁺The ion fragment of the compound (y 2+ Ca-2H) was found to be bound to His as a calcium ion (m/z 192.2381)]⁺(m/z-263.3283) and [ y3+ Ca-2H]⁺(m/z-360.4581) no other sites were bound by calcium ions, so it was determined that one of the binding sites for calcium ions to the peptide was at the His residue; fragment ion [ b2+ Ca-NH3 ]]⁺With fragment ion [ y3+ Ca-2H ]]⁺Indicating that-Pro-does not bind calcium ions, it was determined that another binding site for calcium ions to the peptide was located at Glu residue. As a result of the combined FTIR and mass spectra, the binding reaction of the calcium peptide involved the carboxyl group in the Glu R group with the carboxyl and amino groups on His. Wherein, Glu is acidic amino acid and has stronger metal binding capacity. Notably, His is a basic amino acid, but is also related toCalcium ions undergo a binding reaction, which may be a combined action of His and Ala, since carnosine (His-Ala) has been shown to have a strong metal-binding ability.

The peptide-calcium binding reaction involving the carboxyl group on Glu with the carboxyl and amino groups on His was inferred to bind calcium ions in a bidentate pattern by infrared spectroscopic analysis and mass spectrometric analysis of the peptide-calcium chelate, coupled with the mass-to-charge ratio of the Glu-Pro-ion fragment, in addition, the carboxyl and amino groups on His were bound to calcium ions in an α pattern based on the results of the above structural analysis, a possible peptide-calcium model was constructed using ChemDraw and Chem3D software, see FIG. 21.

In this example, two short peptides having high calcium binding activity were isolated and purified from bonito protein peptide, and identified by Q-TOF as Glu-Pro-Ala-His (MW: 453.3U) and Tyr-Asp-Thr (MW: 398.1U) in their amino acid sequences, respectively, wherein tetrapeptide (Glu-Pro-Ala-His) is a novel calcium binding active peptide which binds calcium ions in such a manner that carboxylic acid groups of Glu bind calcium ions in a bidentate pattern and carboxylic acid groups and amino groups of His chelate calcium ions in a α pattern to form a stable five-ring structure, and based on this result, a hypothetical molecular model of peptide-calcium chelate was constructed.

Example four Effect of Katsuwonus Pelamis protein peptide chelated calcium on bone growth in rats

In the embodiment, by establishing a rat low-calcium model, the influence of the bonito protein peptide chelated calcium with different concentrations on the femoral bone of a rat is analyzed by taking the body length and the weight, the blood calcium and blood phosphorus content, the calcium apparent absorption rate, the bone volume fraction, the bone density and the bone trabecular microstructure of the rat as indexes, and the bioavailability of the bonito protein peptide chelated calcium in an organism is tested.

60 male rats with the age of 4 weeks of weaning are selected and are adaptively raised for 3 days, and free feed is provided for basal maintenance feed and drinking distilled water. Then divided randomly by weight into 6 groups of 10. The breeding environmental conditions are as follows: the temperature is 20 +/-1) DEG C, the relative humidity is 60 +/-5 percent, and the light and the dark alternate period is maintained for 12h/12 h. The feeding period is 5 weeks. The low-calcium feed is prepared according to the formula of 'health food inspection and evaluation technical specification'. Specific grouping and feeding conditions are shown in table 4.

TABLE 4 rat grouping and feeding conditions

Note: the intake dosage calculation is 5, 10 and 20 times of the recommended daily intake (800-1200 mg/60kg) of calcium for adults, and is converted according to the calcium content (15%) of the peptide calcium chelate.

1. Influence on weight and body length of rat

The effect of bonito protein peptide calcium chelate on the weight and height of rats is shown in table 5, and the weight and height of each group of rats are increased. The weight gain rate of each experimental group is higher than that of the blank group, wherein the weight gain rate of the high-dose group is the highest, and the weight gain rates of the medium-dose group and the high-dose group are significantly different from those of the calcium carbonate control group. The growth rate of the body length of each group of rats has no significant difference. This shows that the bonito protein peptide chelated calcium has the effect of promoting the growth of rats.

TABLE 5 influence of Katsuwonus Pelamis protein peptide chelated calcium on weight and height of rat

2. Influence on the apparent absorption rate of calcium

The effect of bonito protein peptide chelated calcium on the apparent absorption rate of calcium in rats is shown in fig. 22. The blank group showed the highest apparent absorption rate of calcium, probably because the rats stimulated the absorption of calcium ions in the intestinal tract in case of severe calcium deficiency, thereby increasing the absorption rate of calcium ions. The apparent absorption rates of the low, medium and high dose groups are not significantly different and are higher than those of the peptide + calcium control group and the calcium carbonate control group, and compared with the calcium carbonate control group, the peptide + calcium control group has better calcium apparent absorption rate, which is related to the existence form of calcium in intestinal tracts. Calcium in the peptide chelated calcium dose group is directly absorbed by intestinal tract in a whole molecule form through a small peptide active transportation way, so that the absorption rate of the calcium is highest. The apparent absorption rate of calcium of the peptide + calcium control group was higher than that of the calcium carbonate control group, indicating that the bonito protein peptide had the effect of promoting the absorption of calcium by rats.

3. Influence on blood calcium concentration, blood phosphorus concentration and A L P Activity in rats

The blood calcium concentration, the blood phosphorus concentration and the A L P activity of rats are affected by the bonito protein peptide chelated calcium, and the results are shown in table 6. the blood calcium concentration and the blood phosphorus concentration of the rats in each experimental group are not significantly different and are all within a normal concentration range, which indicates that calcium supplies in each experimental group do not cause the abnormality of the calcium concentration and the phosphorus concentration in blood.

TABLE 6 influence of Katsuwonus Pelamis protein peptide chelated calcium on blood calcium concentration, blood phosphorus concentration and A L P activity of rat

Note: denotes P < 0.05 for each dose group compared to the blank group.

4. Effect on bone growth in rats

The rat femur consists of cancellous bone, cortical bone and a medullary cavity, wherein the cortical bone has stronger pressure resistance and is distributed on the surface of the bone to play a main supporting role. Cancellous bone is an extension of cortical bone and is composed of interwoven trabeculae of bone. In a calcium deficiency model in rats, bone growth and development levels are often characterized by various morphological characteristics of the cancellous and cortical bone of the femur of the rat. It can be seen from fig. 23-28 that, with the increase of the gavage dosage of bonito protein peptide chelated calcium, the cancellous bone mass of rat femur gradually increases, the cortical bone layer gradually thickens, and it is obviously seen that the bone growth of rat femur in the high dosage group is superior to that in the blank group and the two control groups, which indicates that the peptide chelated calcium has an important effect on promoting the bone growth of rat, and the effect is more obvious when the dosage is higher.

5. Effect on rat bone volume fraction and bone Density

The bone volume fraction (BV/TV) is a common index for evaluating the bone mass of cancellous bone and cortical bone, and can directly reflect the change of the bone mass. As can be seen from Table 7, the bone volume fraction was improved to various degrees in each experimental group compared to the blank group, wherein the BV/TV values of the high dose group and the peptide + calcium control group were 36.29% and 31.23%, respectively, which were significantly improved (P < 0.05%) compared to the blank group (21.17%).

The bone mineral density is called bone density for short, is closely related to the bone strength and the stability of the internal structure of the bone, and is one of important detection indexes for evaluating a low-calcium model and an osteoporosis model. As can be seen from Table 7, the bone density of the rats in each experimental group was increased to various degrees compared to the blank group. Wherein, the cortical bone density of the medium and high dose groups and the peptide + calcium control group is obviously improved compared with the blank group (P is less than 0.05), and the cancellous bone density of the high dose group and the peptide + calcium control group is obviously improved compared with the blank group (P is less than 0.05). The results show that the bonito protein peptide and the peptide calcium chelate have the effect of improving the bone density of rats.

TABLE 7 influence of Katsuwonus Pelamis protein peptide chelated calcium on rat femur BV/TV and BMD

Note: p < 0.05 for each experimental group compared to the calcium carbonate control group, and P < 0.05 for each experimental group compared to the blank group.

6. Effect on rat trabecular bone microstructure

The trabecular bone number (tb.n), trabecular bone thickness (tb.th) and trabecular bone separation (tb.sp) are the main indicators for evaluating the trabecular bone microstructure. The influence of the bonito protein peptide chelated calcium on the bone trabecular microstructure of the rats is shown in table 8, compared with the blank group, the number of the bone trabeculae of the medium-high dose group and the calcium carbonate control group is obviously increased (P is less than 0.05), the separation degree of the bone trabeculae of the high dose group and the peptide + calcium group is obviously reduced (P is less than 0.05), and the thickness of the bone trabeculae of the thighbone of the rats of each group is not obviously different (P is less than 0.05). The results of this experiment were consistent with the results for bone mass counts and bone density.

Table 8 influence of bonito protein peptide chelated calcium in platyphylus skipjack on rat bone trabecular microstructure

Note: denotes P < 0.05 for each dose group compared to the blank group.

The analysis results of the bone volume fraction, the bone density and the bone trabecula microstructure show that the bonito protein peptide chelated calcium can improve the bioavailability of calcium in a rat body, increase the precipitation of the calcium in bones and promote the growth and development of the bones.

The bonito protein peptide chelated calcium can improve the volume fraction of rat bones, increase the bone density of rats and improve the microstructure of bone trabeculae, promote the growth and development of bones, and after the bonito protein peptide chelated calcium is supplemented to the calcium-deficient rats, the integral fraction is improved to 36.29 percent, the bone densities of cortical bones and cancellous bones are respectively 6303.39mg/cc and 4126.09mg/cc, and the number of the bone trabeculae is 4.87mm^-1The increase was significant compared to the calcium carbonate control.

The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A preparation method of peptide calcium chelate comprises the steps of carrying out a chelating reaction of a protein source and a calcium source in water, wherein the protein source is selected from an Auxis thazard protein peptide; setting the parameters of the chelation reaction: the mass ratio of the protein source to the calcium source is 1: 3-3: 1, the chelation temperature is 30-70 ℃, the chelation time is 10-50 min, and the pH value is 7-11.

2. The method for producing a peptide-calcium chelate according to claim 1, wherein the bonito protein peptide is produced by subjecting bonito to enzymatic hydrolysis with a protease; the protease is selected from papain and flavourzyme; and (3) carrying out enzyme deactivation on the enzymolysis product, then centrifuging, deoiling and decoloring the supernatant, filtering by using a ceramic membrane and an ultrafiltration membrane, and concentrating and drying to obtain the bonito protein peptide with the molecular weight not higher than 5000U.

3. The method for producing a peptide-calcium chelate according to claim 2, wherein the method for producing a bonito protein peptide comprises:

4. The method for preparing peptide calcium chelate according to claim 1, wherein the parameters of the chelation reaction are as follows: the mass ratio of the protein source to the calcium source is 2:1, the chelation temperature is 50 ℃, the chelation time is 20min, and the pH value is 9.

5. The method for producing a peptide-calcium chelate according to claim 1, wherein the bonito protein peptide contains calcium-binding active peptides Glu-Pro-Ala-His and Tyr-Asp-Thr.

6. The method for preparing a peptide calcium chelate complex according to claim 5, wherein the Glu-Pro-Ala-His is bound to calcium ions in such a manner that the carboxylic acid group of Glu binds to calcium ions in a bidentate mode and the carboxylic acid group and the amino group of His are chelated to calcium ions in an α mode to form a five-ring structure.

7. A peptide calcium chelate prepared using the method of any one of claims 1-6.

8. A protein peptide derived from Katsuwonus Pelamis (Auxis thazard), the protein peptide comprising a peptide having Glu-Pro-Ala-His and Tyr-Asp-Thr, which are calcium-binding active peptides;

9. The application of a protein peptide in preparing a peptide calcium chelate, wherein the protein peptide is a bonito (Auxisthazard) protein peptide.

10. The use of a peptide calcium chelate or a medicament containing the peptide calcium chelate for promoting bone growth and development.