Jellyfish collagen combined calcium and preparation method thereof

Abstract

The invention relates to the technical field of functional health food, and discloses a preparation method for crosslinking calcium ions and collagen, and the product can be used as a nutritional supplement for preventing and improving osteoporosis. According to the invention, jellyfish collagen and succinic anhydride are firstly acylated, and the acylated jellyfish collagen keeps a triple-helical structure without change and carries more negative charges, so that the jellyfish collagen is combined with calcium ions through coulomb interaction, and the content of the calcium ions in the collagen can be increased to 2.34 * 104 mg / kg from 127 mg / kg. The invention provides a product capable of simultaneously supplementing calcium and collagen in a brand new manner, so that calcium ions can be better absorbed in intestinal tracts and are deposited in bones, the bone toughness is enhanced, the bone density is also improved, and the effects of preventing and relieving osteoporosis are achieved.

Description

Technical Field

[0001] The invention relates to the technical field of functional health food, in particular to jellyfish collagen combined with calcium and a preparation method thereof. Background Art

[0002] Calcium is one of the most abundant nutrients in the human body and plays an extremely important role in human life activities. Insufficient calcium intake can lead to osteoporosis, rickets, osteomalacia and other diseases. The bone structure consists of two parts, the inorganic part of the bone (accounting for about 75% of the dry bone weight) and the organic part (accounting for about 25% of the dry bone weight). The inorganic part is mainly hydroxyapatite crystals mainly composed of calcium. 90-95% of the organic part is collagen. The collagen matrix provides strength and bone elasticity. Collagen can increase bone collagen synthesis through absorption and induce osteoblast differentiation of bone marrow cells. Therefore, the best way to supplement calcium is to supplement collagen combined with calcium in time. Collagen can effectively absorb calcium, so that calcium is deposited in the bones instead of deposited in other parts to form stones.

[0003] Commonly used calcium supplements include inorganic calcium, organic calcium and chelated calcium. Inorganic calcium must be absorbed with the help of human gastric acid and related enzymes, and vitamins as carriers. Even if inorganic calcium is dissociated into calcium ions, it is difficult to bind to calcium-binding proteins due to lack of affinity, and has the disadvantages of low absorption rate and low calcium content. Although organic calcium is easy to dissociate into ionic calcium, calcium ions are easily combined with gastric acid and acidic substances such as oxalic acid and phytic acid in food in the stomach to form precipitations such as calcium oxalate and calcium phytate, which hinder the absorption of calcium, so the absorption rate of organic calcium is also relatively low. Peptide calcium chelates have attracted much attention as a new generation of calcium supplements. Their advantages are that they are less irritating to the stomach and intestines, and are easier to penetrate biological membranes and be absorbed and utilized by the human body, overcoming the shortcomings of inorganic calcium and organic calcium. However, peptide calcium chelates are easily overdigested under the action of gastric acid and enzymes. Since calcium ions cannot provide empty orbitals for forming coordination bonds, the binding of peptides and calcium ions is essentially just an electrostatic interaction, and no stable coordination bonds are formed. It is very easy to cause their structure to be destroyed during the digestion and absorption process of the human body. In addition, although peptide calcium chelates are easily absorbed by the intestines, the sharp increase in blood calcium and blood peptides in a short period of time is often excreted from the body before it can be absorbed and is not fully utilized by the human body.

[0004] The prevention and treatment of osteoporosis cannot be completely solved by simply supplementing calcium. A high calcium absorption rate must be supplemented with a high calcium deposition and a high utilization rate to fundamentally solve the problem. Sufficient calcium intake and corresponding collagen are important conditions for osteogenesis, and both are indispensable. They can fully and effectively increase bone density, enhance bone elasticity and toughness, and effectively prevent osteoporosis.

[0005] The Chinese patent "A collagen-calcium chelate and its preparation method (CN102178228A)" discloses a chelate and its preparation method using animal-derived collagen or human-like collagen as raw materials and combined with a soluble calcium salt. Some physical and chemical properties of the chelate and its efficacy evaluation were studied, but the key parameter of the chelate, the calcium content, was not monitored and compared, which has certain limitations. Xu Zhou et al. (Document: "The Effect of Succinylation on the Bioactive Structure of Collagen" Journal of Food Science, Volume 37, Pages 12-16, etc.) took pig skin collagen as the research object, prepared acylated collagen by modification with succinic anhydride, and investigated its various properties. The results showed that acylation would not destroy the triple helix structure of collagen and its biological activity. However, Xu et al. did not study the calcification of the acylated collagen. Summary of the invention

[0006] 1. Technical issues to be resolved

[0007] In view of the deficiencies of the prior art, the present invention provides a jellyfish collagen combined with calcium and a preparation method thereof, which allows the collagen to combine with more calcium while maintaining its triple helix structure. Even after gastrointestinal digestion, it can still be absorbed by the human body in the form of peptide segments, retaining some of the activity of collagen, making it easier to be recognized by cells, thereby allowing calcium and collagen to be deposited in the bones at the same time, enhancing the toughness and hardness of the bones. This solves the problem of poor absorption and low utilization rate of calcium supplements on the existing market, and effectively improves osteoporosis from the two dimensions of calcium supplementation and collagen supplementation.

[0008] (II) Technical solution

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] Preferably, an appropriate amount of jellyfish collagen solid is taken to prepare a collagen suspension of a certain concentration. Furthermore, the selected jellyfish collagen is a macromolecular collagen (relative molecular mass of more than 300 kDa) extracted from jellyfish, having a triple helix structure, and is prepared into a suspension containing 1% to 10% jellyfish collagen with deionized water;

[0011] Preferably, succinic anhydride is slowly added at 0°C, the pH is adjusted, and the reaction is stirred for a period of time to obtain acylated collagen. Furthermore, the mass of succinic anhydride added is 10% of the mass of collagen; since acetic acid is produced as a byproduct during the reaction, the pH needs to be repeatedly adjusted with sodium hydroxide to control the pH of the reaction system at about 9.0, and the reaction time is 4 hours.

[0012] Preferably, the pH of the acylated collagen suspension is adjusted, calcium chloride is added, stirred to dissolve, and then allowed to stand at 0° C. to 10° C. for reaction. Furthermore, the mass ratio of collagen to calcium chloride is 2:1, the pH is adjusted to 5.3 to 5.8, and the soaking time is 1 to 32 hours, preferably 24 hours.

[0013] Preferably, 1 mol / L sodium chloride is added for salting out for 24 hours, and centrifuged at 10000 r / min for 30 min to obtain a precipitate.

[0014] Preferably, the precipitate is redissolved in 0.05 mol / L glacial acetic acid, dialyzed in 0.1 mol / L glacial acetic acid solution for 24 hours, and then dialyzed in deionized water for 48 hours. The dialyzed sample is freeze-dried to obtain a product of jellyfish collagen bound to calcium.

[0015] (III) Beneficial effects

[0016] Compared with the prior art, the present invention provides a jellyfish collagen-bound calcium and a preparation method thereof, which has the following beneficial effects:

[0017] 1. The invention provides a jellyfish collagen combined with calcium, which has the advantages of high calcium content and is easily absorbed and utilized by the human body. It can deposit calcium and collagen in the bones at the same time, enhance the toughness and hardness of the bones, improve the symptoms of osteoporosis and promote physical health.

[0018] 2. The preparation method provided by the invention introduces carboxyl groups into jellyfish collagen through acylation reaction with succinic anhydride, so that it has a negative charge and can bind more calcium ions, thereby increasing the calcium ion content in collagen from 127 mg / kg to 2.34×10 4 mg / kg.

[0019] 3. The jellyfish collagen combined with calcium provided by the invention adopts large-molecule collagen as raw material, and the digestion and absorption period of the collagen in the human body is about 1-4 hours. Compared with small-molecule peptides, it can continuously provide calcium and peptides into the blood, avoiding the waste caused by untimely absorption due to sudden increase in blood calcium and blood peptide concentrations, which is truly beneficial to the absorption and utilization by the human body.

[0020] 4. The preparation method provided by the invention retains the original structure and activity of jellyfish collagen. While supplementing calcium, it also has the effects of improving immunity, lowering blood pressure and blood lipids, and scavenging free radicals.

[0021] 5. The preparation method provided by the invention has mild reaction, low cost, simple and easy operation, no pollution to the environment, and can be suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1is the calcium content of the jellyfish collagen, the products in Example 1 and Comparative Example 1;

[0023] Figure 2 is the calcium content of the jellyfish collagen, the products in Example 1 and Comparative Example 2;

[0024] Figure 3 is the calcium content in the jellyfish collagen, the products of Example 1 and Comparative Example 3;

[0025] Figure 4 is the calcium content in the jellyfish collagen, the products of Example 1 and Comparative Example 4;

[0026] Figure 5 It is the UV spectrum of the product of Example 1;

[0027] Figure 6 The SDS-PAGE gel electrophoresis diagram of the product of Example 1;

[0028] Figure 7 Circular dichroism spectrum of the product of Example 1. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Example 1

[0031] Step S1: Sol

[0032] Weigh 500 mg of jellyfish collagen and dissolve it in 50 mL of deionized water to form a suspension.

[0033] Step S2: Acylation

[0034] 50 mg of succinic anhydride was added, and the pH was adjusted with 1 mol / L sodium hydroxide, and the pH of the reaction system was kept at about 9.0 and stirred at 0°C for 4 hours.

[0035] Step S3: Calcium cross-linking

[0036] Adjust the pH to 5.5 with 0.1 mol / L sodium hydroxide solution, add 250 mg of calcium chloride, and soak at 4°C for 24 hours.

[0037] Step S4: Salting out centrifugation

[0038] Add 1 mol / L sodium chloride for salting out for 24 hours, centrifuge at 10000 r / min for 30 min, and take the precipitate.

[0039] Step S5: Dialysis and drying

[0040] The precipitate was redissolved in 0.05 mol / L glacial acetic acid, dialyzed in 0.1 mol / L glacial acetic acid solution for 24 hours, and then dialyzed in deionized water for 48 hours, with the solution changed every 8 hours. The dialyzed sample was freeze-dried to obtain a product of jellyfish collagen bound to calcium.

[0041] The final calcium content of this example was determined by atomic absorption spectrometry to be 2.34×10 4 mg / kg.

[0042] Comparative Example 1

[0043] The difference between this comparative example and Example 1 is that, in step S2: acylation, the amount of succinic anhydride added is 25 mg (5% of the mass of jellyfish collagen), 75 mg (15% of the mass of jellyfish collagen), and 100 mg (20% of the mass of jellyfish collagen), respectively. The remaining steps and conditions of this comparative example are the same as those of Example 1.

[0044] The final calcium content of this comparative example was determined by atomic absorption spectrometry to be 1.97×10 4 mg / kg, 2.29×10 4 mg / kg, 2.05×10 4 mg / kg.

[0045] Comparative Example 2

[0046] The difference between this comparative example and Example 1 is that, in step S3: calcium cross-linking, the immersion temperatures are 0° C. and 8° C. respectively. The remaining steps and conditions of this comparative example are the same as those of Example 1.

[0047] The final calcium content of this comparative example was determined by atomic absorption spectrometry to be 1.89×10 4 mg / kg, 2.11×10 4 mg / kg.

[0048] Comparative Example 3

[0049] The difference between this comparative example and Example 1 is that, in step S3: calcium cross-linking, the immersion time is 4, 8, 16 and 32 hours respectively. The remaining steps and conditions of this comparative example are the same as those of Example 1.

[0050] The final calcium content of this comparative example was determined by atomic absorption spectrometry to be 1.4×10 4 mg / kg, 1.56×10 4 mg / kg, 2.16×104 mg / kg, 2.17×10 4 mg / kg.

[0051] Comparative Example 4

[0052] The difference between this comparative example and Example 1 is that, in step S3: calcium cross-linking, the pH is adjusted to 4.0, 4.5, 5.0, 6.0, and 6.5 respectively. The remaining steps and conditions of this comparative example are the same as those of Example 1.

[0053] The final calcium content of this comparative example was determined by atomic absorption spectrometry to be 1.81×10 4 mg / kg, 2.02×10 4 mg / kg, 2.25×10 4 mg / kg, 2.29×10 4 mg / kg, 2.17×10 4 mg / kg.

[0054] The product of Example 1 was subjected to UV spectroscopy analysis, SDS-PAGE gel electrophoresis analysis and circular dichroism analysis, and the analysis results are as follows:

[0055] 1. Ultraviolet spectrum analysis of the product of Example 1

[0056] Figure 5 It is shown in the figure that the maximum absorption peak of the product of Example 1 coincides with the maximum absorption peak of jellyfish collagen (λ=230 nm), indicating that the conjugated system of jellyfish collagen does not change after acylation and binding to calcium.

[0057] 2. SDS-PAGE gel electrophoresis analysis of the product of Example 1

[0058] from Figure 6 Through the analysis of Lane A and Lane B, it can be seen that the relative molecular mass distribution range of the product of Example 1 is widened, and the molecular weight is greater than that of jellyfish collagen. This is mainly because the large molecular weight jellyfish collagen is not destroyed, and the increase in succinyl groups is manifested as an increase in molecular weight.

[0059] 3. Circular dichroism analysis of the product of Example 1

[0060] from Figure 7 From the circular dichroism spectrum, it can be seen that the product of Example 1 has a negative absorption peak at about 196nm, also known as the negative Cotton effect, and a weak positive absorption peak at about 203nm, namely the positive Cotton effect. This is consistent with the characteristics of jellyfish collagen, indicating that the product of Example 1 still maintains a good triple helix structure.

[0061] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing jellyfish collagen combined with calcium, characterized in that: The following steps are involved: (1) Sol: Take an appropriate amount of jellyfish collagen solid and prepare a collagen suspension of a certain concentration; (2) Acylation: Succinic anhydride is slowly added at a certain temperature, the pH is adjusted, and the reaction is stirred for a period of time to obtain acylated collagen; (3) Calcium cross-linking: Add calcium chloride to the acylated collagen, stir to dissolve, adjust the pH and let it stand to react; (4) Separation by precipitation: salt out with sodium chloride overnight and centrifuge to obtain the precipitate; (5) Dialysis and drying: The precipitate was redissolved in 0.05 mol / L glacial acetic acid, dialyzed in 0.1 mol / L glacial acetic acid solution for 24 h, and then dialyzed in deionized water for 48 h. The dialyzed sample was freeze-dried to obtain a product of jellyfish collagen bound to calcium.

2. The jellyfish collagen-bound calcium and preparation method thereof according to claim 1, characterized in that: The collagen described in step (1) is a macromolecular collagen (relative molecular weight of 300 kDa or more) extracted from jellyfish, has a triple helix structure, and is prepared with deionized water into a suspension containing 1% to 10% jellyfish collagen.

3. The jellyfish collagen-bound calcium and preparation method thereof according to claim 1, characterized in that: The reaction temperature in step (2) is 0°C to 4°C; the mass of succinic anhydride added is 0% to 30% of the mass of collagen; since acetic acid is produced as a byproduct during the reaction, the pH needs to be repeatedly adjusted with sodium hydroxide to keep the pH of the reaction system in the range of 4 to 9; and the reaction time is controlled to be 4 to 6 hours.

4. The jellyfish collagen-bound calcium and the preparation method thereof according to claim 1, characterized in that: The reaction temperature in step (3) is 0°C to 10°C, the mass ratio of collagen to calcium chloride is 2:1, the pH is adjusted to 4.0 to 6.5, and the soaking time is 1 to 32 hours.

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