Chlorella pyrenoidosa peptide calcium chelate and preparation method thereof

Abstract

The invention provides a chlorella pyrenoidosa calcium chelating peptide and a chlorella pyrenoidosa peptide calcium chelate prepared by using the chlorella pyrenoidosa calcium chelating peptide. The prepared chlorella pyrenoidosa peptide calcium chelate can enhance the maximum load and structural strength of the tibia of a mouse with osteoporosis. The chlorella pyrenoidosa calcium chelating peptide provided by the invention is combined with calcium ions to form a peptide-calcium chelate, and animal experiments prove that the peptide-calcium chelate can significantly improve the maximum load and structural strength of the tibia of a mouse with osteoporosis, and has a good effect of improving the bone biomechanical properties of the mouse with osteoporosis.

Description

Technical Field

[0001] The invention belongs to the technical field of biologically active product processing, and in particular relates to a chlorella protein core peptide calcium chelate and a preparation method thereof. Background Art

[0002] Calcium is one of the most common inorganic elements in the human body and an essential nutrient for the human body. It plays a key role in biological activities such as metabolism, muscle contraction, and signal transmission. With the increase of age, the loss of bone calcium becomes more and more serious, especially after the age of 65, women lose about 30%-50% of bone calcium. Calcium deficiency can cause a variety of diseases, such as osteoporosis, hypertension, kidney stones, etc. Existing calcium supplements such as inorganic calcium, organic calcium, and amino acid calcium provide sufficient calcium content, but ignore the body's absorption of calcium.

[0003] Daily diet is the main way to ingest calcium, but in the alkaline environment of the intestine, calcium in ionic form easily forms insoluble precipitates with phosphates, etc., thereby reducing its absorption efficiency and bioavailability. As the fourth generation of calcium supplements, peptides have abundant calcium binding sites and can be well absorbed by the small intestine. The source of calcium chelating peptides mainly has two criteria: (1) the value-added utilization of a large amount of underutilized proteins or protein-rich food industry by-products; (2) the utilization of proteins containing specific peptide sequences or amino acid residues that are responsible for binding calcium. Proteins from other food sources contain a large number of new sequences with potential calcium chelating activity that have not yet been discovered in their primary structures. In recent years, peptide calcium chelates prepared by combining calcium ions with food-derived calcium chelating peptides have become a research hotspot. Peptide calcium chelates not only significantly enhance calcium absorption and utilization, but also exhibit various biological activities, which provides a new way to solve human calcium deficiency.

[0004] Currently, the global consumer demand for protein is gradually increasing. The marine environment can provide a variety of biological resources. Due to its strong tolerance to extreme and competitive environments, high nutritional value, low price and high yield, microalgae have become a potential source of edible protein and bioactive compounds. Chlorella pyrenoidosa is a potential source for the preparation of calcium chelating peptides because it contains rich protein and amino acids with strong calcium binding ability such as glutamic acid and aspartic acid. Therefore, it is of great research significance and economic value to study calcium chelating peptides with high calcium chelating activity from Chlorella pyrenoidosa and prepare peptide calcium chelates. Summary of the invention

[0005] The invention provides a pyrenoid chlorella calcium chelate peptide and a preparation method thereof, and a pyrenoid chlorella peptide calcium chelate and a preparation method thereof. The prepared pyrenoid chlorella peptide calcium chelate can enhance the maximum load and structural strength of the tibia of osteoporotic mice.

[0006] The present invention first provides a pyrenoid chlorella calcium chelating peptide, and the preparation method thereof is as follows:

[0007] 1) Broken cell wall of Chlorella pyrenoidosa

[0008] The pyrenoid chlorella and water are fully mixed, and the cell wall breaking operations of repeated freezing and thawing, high temperature and high pressure, and high pressure homogenization are respectively performed;

[0009] The sufficient mixing is to mix the protein nucleus Chlorella and water in a ratio of 1:10 (w / v), use a disperser to beat for 10 minutes, and mix thoroughly; preferably, repeated freezing and thawing is used as the cell wall breaking method, and the repeated freezing and thawing is freezing in a -20°C cold storage for 3-4 times, each time for 10-12 hours, and thawing at room temperature for 3-4 times.

[0010] 2) High temperature and high pressure treatment of Chlorella pyrenoidosa

[0011] The repeatedly frozen and thawed Chlorella vulgaris solution is subjected to high temperature and high pressure treatment; the high temperature and high pressure treatment is 121° C., 0.1 MPa, and the treatment lasts 15-20 minutes.

[0012] 3) Preparation of Chlorella pyrenoidosa calcium chelating peptide

[0013] Protease is added to perform enzymolysis, and the enzyme is inactivated after the enzymolysis is completed. The enzymolysis solution is centrifuged, concentrated, and dried to obtain the protein core Chlorella calcium chelating peptide;

[0014] The protease is alkaline protease, neutral protease, flavor protease, papain;

[0015] Preferably, the protease is alkaline protease and flavor protease;

[0016] The enzymatic hydrolysis is carried out at pH 7-9 and 50-55°C for 4-6 hours;

[0017] Preferably, the enzymatic hydrolysis is performed by adding 3.5-4% alkaline protease and flavor protease in a mass ratio of 3:1 (w / w), and hydrolyzing for 6 hours in a water bath at pH 8 and 50°C.

[0018] The amino acid sequence of the Chlorella calcium chelating peptide is DLEPVPGEEN (SEQ ID NO: 1), DIEPVPGEENQY (SEQ ID NO: 2) or DIEPVPGEE (SEQ ID NO: 3).

[0019] The present invention provides a protein core chlorella peptide calcium chelate, and the preparation method thereof is as follows:

[0020] The pyrenoid chlorella calcium chelate peptide is fully mixed with water to prepare a pyrenoid chlorella calcium chelate peptide solution, and calcium chloride dihydrate is added and mixed, and a chelate reaction is carried out in a water bath. After the reaction is completed, anhydrous ethanol is added, and the pyrenoid chlorella peptide calcium chelate is obtained by centrifugation.

[0021] The pyrenoid chlorella calcium chelate peptide solution is a solution prepared by thoroughly mixing the pyrenoid chlorella calcium chelate peptide obtained by concentration and drying with water, and the mass concentration is 50-60g / L;

[0022] The solution system of the chelation reaction is a mixed solution system of pyrenoid chlorella calcium chelate peptide solution and calcium chloride dihydrate, and the mass ratio of pyrenoid chlorella calcium chelate peptide to calcium ion is 5:1 (w / w);

[0023] Preferably, the chelation reaction is carried out under the conditions of pH 9.5-10.0 and 25-30°C water bath for 20-30 minutes;

[0024] The protein core chlorella peptide calcium chelate is a precipitate obtained by adding eight times the volume of anhydrous ethanol, mixing and standing for 8-12 hours after the chelation reaction is completed.

[0025] The Chlorella pyrenoidosa calcium chelating peptide provided by the present invention can be used for preparing calcium supplement preparations.

[0026] The pyrenoid chlorella calcium chelate peptide provided by the present invention combines with calcium ions to form a peptide calcium chelate, which has been shown in animal experiments to significantly improve the maximum load and structural strength of the tibia of osteoporotic mice and has a good effect of improving the biomechanical properties of the bones of osteoporotic mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The graph shows the calcium chelating activity and polypeptide yield of the alkaline protease hydrolysates of Chlorella vulgaris treated with different cell wall breaking methods.

[0028] Figure 2 The nitrogen recovery rate, hydrolysis degree, calcium chelating activity and molecular weight of Chlorella pyrenoidosa hydrolysate before and after high temperature and high pressure treatment.

[0029] Figure 3 The graph shows the calcium chelating activity and hydrolysis degree of Chlorella pyrenoidosa hydrolysate at different compound enzyme ratios, enzymatic hydrolysis time and enzyme addition amount.

[0030] Figure 4 Diagram for optimization of calcium chelation conditions for Chlorella pyrenoidosa peptide.

[0031] Figure 5 This is a diagram showing the effect of Chlorella protein core peptide calcium chelate on bone biomechanics in osteoporotic mice. DETAILED DESCRIPTION

[0032] The present invention uses a combination of repeated freeze-thaw wall breaking and high temperature and high pressure treatment to pre-treat Chlorella pyrenoidosa and then perform enzymolysis. Compared with the enzymolysis obtained before pre-treatment, the enzymolysis obtained after pre-treatment has higher calcium chelation activity. By optimizing the chelation temperature, chelation time, pH, peptide-calcium ratio and other conditions, a Chlorella pyrenoidosa peptide calcium chelate with a high calcium content is prepared. Animal experiments have shown that a high dose of Chlorella pyrenoidosa peptide calcium chelate can significantly improve the maximum load and structural strength of the tibia of osteoporotic mice.

[0033] The present invention is described in detail below in conjunction with specific embodiments and drawings.

[0034] Example 1: Preparation of Chlorella Peptide

[0035] The preparation of chlorella peptide with high calcium chelating activity comprises the following steps:

[0036] 1) Mix Chlorella vulgaris and water in a ratio of 1:10 (w / v), use a disperser to beat for 10 minutes, mix thoroughly, freeze in a -20°C cold storage for 4 times, each time for 12 hours, and thaw at room temperature for 4 times. The pH of the repeatedly frozen and thawed Chlorella vulgaris solution was adjusted to 6.5 with 0.1M NaOH, and subjected to high temperature and high pressure treatment at 121°C and 0.1MPa for 15 minutes.

[0037] 2) adding alkaline protease and flavor protease in a mass ratio of 3:1 (w / w), adding 4% enzyme, adjusting the pH value of the system to 8.0, keeping the system hydrolyzed at 50°C for 6 hours, boiling and killing the enzyme after the enzymatic hydrolysis, centrifuging the enzymatic hydrolyzate, concentrating and drying to obtain protein core Chlorella calcium chelating peptide;

[0038] In this example, the cell wall of Chlorella vulgaris was broken by repeated freezing and thawing, high temperature and high pressure, and high pressure homogenization, and then enzymatic hydrolysis was performed with 4% alkaline protease to obtain enzymatic hydrolysis products. Figure 1 As shown in the figure, the peptide yield of the alkaline protease hydrolysate of Chlorella vulgaris after repeated freeze-thaw wall-breaking treatment is not as good as that of the hydrolysates treated by the other two wall-breaking methods, but its calcium chelation activity is significantly higher than that of the hydrolysates treated by the other two wall-breaking methods, indicating that the repeated freeze-thaw method can produce more peptides with calcium chelation activity. After the repeatedly frozen-thawed Chlorella vulgaris was treated with high temperature and high pressure, four enzymes, alkaline protease, neutral protease, papain and flavor protease, were selected for enzymolysis to obtain enzymolysis products. The results are shown in the figure. Figure 2 As shown in the figure, after high temperature and high pressure treatment, except for papain hydrolysate, the nitrogen recovery rates of the other three hydrolysates were significantly improved, the hydrolysis degrees of the four hydrolysates were all decreased, the calcium chelating activity of the flavor protease hydrolysate was significantly improved, and the part of the protein core Chlorella hydrolysate prepared by high temperature and high pressure treatment with a molecular weight less than 500Da accounted for a large proportion.

[0039] Alkaline protease and flavor protease with high calcium chelating activity were selected for compound enzymatic hydrolysis. The effects of different compound enzyme ratios, enzymatic hydrolysis time, and enzyme addition amount on calcium chelating activity were explored through single factor experiments. The results are as follows: Figure 3 As shown, when the mass ratio of flavor protease to alkaline protease is 1:3 (w / w), the enzymolysis time is 6h, and the amount of enzyme added is 5%, the calcium chelating activity of the enzymolyzate is high. According to the results of the single factor experiment, a three-factor three-level orthogonal experiment was performed to optimize the enzymolysis process. As shown in Table 1, the best enzymolysis process for the protein core chlorella peptide with high calcium chelating activity is the mass ratio of flavor protease to alkaline protease of 1:3 (w / w), the enzymolysis time is 6h, and the amount of enzyme added is 4%. After verification, the calcium binding capacity of the protein core chlorella peptide prepared in this embodiment is 13.2039±1.6304μg / mg.

[0040] Table 1: Orthogonal experiment table of enzymatic hydrolysis process

[0041]

[0042] The peptide fingerprint of the pyrenoidosa calcium chelating peptide was determined by high-resolution mass spectrometry. The pyrenoidosa protein database was added and the library was searched using Proteome Discoverer. A total of 560 peptides were matched. Studies have shown that acidic amino acids have better calcium binding ability due to their two carboxyl groups. Therefore, peptide sequences containing two or more acidic amino acids were screened out, as shown in Table 2.

[0043] Table 2: Sequence Listing of Chlorella pyrenoidosa Calcium Chelating Peptides

[0044]

[0045] Peptides with amino acid sequences of DLEPVPGEEN, DIEPVPGEENQY, and DIEPVPGEE were synthesized and their calcium binding ability was verified. The results showed that all three peptides had high calcium chelating ability.

[0046] Example 2: Preparation of Chlorella Peptide Calcium Chelate

[0047] The protein-nuclear Chlorella calcium chelate peptide was added into water to make its mass concentration 50 g / L, and then calcium ions with a peptide-calcium mass ratio of 5:1 (w / w) were added, stirred evenly, and bathed in water at pH 10 and 30°C for 30 minutes, and eight times the volume of anhydrous ethanol was added, stirred evenly and allowed to stand for 12 hours, and precipitated by centrifugation, and the protein-nuclear Chlorella peptide calcium chelate was obtained by drying.

[0048] In this example, the calcium chelation conditions of the protein core chlorella peptide were optimized, and the results were as follows: Figure 4As shown, four factors, namely chelation temperature, chelation time, peptide calcium mass ratio and pH, were selected for single factor experiments, and the optimal conditions for protein core chlorella peptide calcium chelation were obtained as peptide calcium mass ratio of 5:1 (w / w), pH of 10, chelation temperature of 30℃, and chelation time of 30min. After verification, the calcium content of protein core chlorella peptide calcium chelate prepared by this process was 192.0038±7.9082μg / mg.

[0049] Example 3: Detection of the effect of pyrenoid chlorella peptide calcium chelate on improving bone biomechanical properties

[0050] The present invention establishes an ovariectomized osteoporosis mouse model, and intragastric administration of pyrenoid chlorella peptide calcium chelate to the mice, and the specific steps are as follows:

[0051] 1. Animal husbandry and experimental design

[0052] C57BL / 6J female mice were randomly divided into a sham operation group and an ovariectomy group. The ovariectomy group had bilateral ovaries removed, while the sham operation group had only a small amount of fat tissue removed after opening the abdominal cavity. Samples were gavaged one week after surgery and continued for 12 weeks. The specific groups were as follows: ① Sham operation group (SHAM, n = 6): pure water was gavaged at a dose of 0.2 mL per 20 g of mouse body weight per day; ② Model group (MC, n = 6): pure water was gavaged at a dose of 0.2 mL per 20 g of mouse body weight per day; ③ Positive control group (ALN, n = 6): alendronate sodium was gavaged at 1 mg / kg; ④ Calcium carbonate group (CaCO 3 , n = 6): intragastric administration of calcium carbonate (Ca 2+ ⑤ Low-dose chlorella peptide calcium chelate group (CCL, n = 6): intragastric administration of chlorella peptide calcium chelate (Ca 2+ ⑥ High-dose chlorella peptide calcium chelate group (CCH, n = 6): intragastric administration of chlorella peptide calcium chelate (Ca 2+ The dose is 104 mg / kg / d).

[0053] 2. Animal materials

[0054] After 12 weeks of intragastric administration, the mice were sampled. They were fasted for 12 hours before sampling. After blood was collected from the eyeballs, the mice were killed by dislocation of the neck. The left and right tibiae were taken and fixed in 4% neutral formaldehyde fixative. The bone biomechanics of the mouse tibiae were measured using a small animal bone strength tester. Figure 5As shown in the figure, the maximum load and structural strength of the MC group were significantly lower than those of the SHAM group, indicating that osteoporosis modeling was successful and the biomechanical properties of mouse bones decreased. The maximum load and structural strength of the CCH group were 1.739±0.386N and 2.074±0.242N, respectively, which were 34.91% and 33.03% higher than those of the MC group.

[0055] The results show that the protein core chlorella peptide calcium chelate prepared by the present invention can improve the bone biomechanical properties of osteoporotic mice and reduce the risk of fractures.

Claims

1. A pyrenoid chlorella calcium chelating peptide, characterized in that: The preparation method of the pyrenoid chlorella calcium chelating peptide is as follows: 1) Broken cell wall of Chlorella pyrenoidosa Mixing Chlorella pyrenoidosa with water, and freezing and thawing to obtain a Chlorella pyrenoidosa solution; 2) High temperature and high pressure treatment of Chlorella pyrenoidosa The frozen-thawed Chlorella pyrenoidosa solution is subjected to high temperature and high pressure treatment; 3) Preparation of Chlorella pyrenoidosa calcium chelating peptide The proteinase is added to the Chlorella vulgaris treated with high temperature and high pressure for enzymolysis, and the enzyme is inactivated after the enzymolysis is completed. The enzymolysis solution is centrifuged and concentrated and dried to obtain the Chlorella vulgaris calcium chelating peptide; the protease is any one or more of alkaline protease, neutral protease, flavor protease and papain.

2. The pyrenoid chlorella calcium chelating peptide according to claim 1, characterized in that The high temperature and high pressure treatment in 2) is 121° C., 0.1 MPa, and the treatment lasts 15-20 minutes.

3. The Chlorella calcium chelating peptide according to claim 1, characterized in that: The adding of protease for enzymolysis in 3) is adding alkaline protease and flavor protease for enzymolysis.

4. The Chlorella calcium chelating peptide according to claim 1, characterized in that: The enzymatic hydrolysis is to add alkaline protease and flavor protease with a concentration of 3.5-4%, and then hydrolyze for 6 hours under the conditions of pH 8 and 50°C water bath; wherein the mass ratio of alkaline protease to flavor protease is 3:

1.

5. The Chlorella pyrenoidosa calcium chelating peptide according to claim 1, characterized in that: The amino acid sequence of the pyrenoid chlorella calcium chelating peptide is DLEPVPGEEN, DIEPVPGEENQY or DIEPVPGEE.

6. A protein core chlorella peptide calcium chelate, characterized in that: The preparation method of the protein core chlorella peptide calcium chelate is as follows: The pyrenoid chlorella calcium chelate peptide according to any one of claims 1 to 5 is mixed with water to prepare a pyrenoid chlorella calcium chelate peptide solution, and then calcium chloride dihydrate is added, mixed, and a chelate reaction is carried out in a water bath. After the reaction is completed, anhydrous ethanol is added, and centrifugation is performed to obtain a pyrenoid chlorella peptide calcium chelate.

7. The pyrenoid chlorella peptide calcium chelate according to claim 6, characterized in that: The mass concentration of the Chlorella pyrenoidosa calcium chelating peptide solution is 50-60 g / L; the mass ratio of the Chlorella pyrenoidosa calcium chelating peptide to calcium ions is 5:

1.

8. The pyrenoid chlorella peptide calcium chelate according to claim 6, characterized in that: The chelation reaction is carried out under the conditions of pH 9.5-10.0 and 25-30° C. water bath for 20-30 minutes.

9. Use of the pyrenoid chlorella peptide calcium chelate according to claim 6 in the preparation of calcium supplement preparations.

10. A calcium supplement preparation, characterized in that: The calcium supplement preparation contains the pyrenoid chlorella peptide calcium chelate according to claim 6 at a pharmacologically effective concentration.

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