A chlorella vulgaris peptide calcium chelate and a method of making the same

Abstract

The present application 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 an osteoporosis mouse. The Chlorella pyrenoidosa calcium-chelating peptide provided by the present application combines with calcium ions to form a peptide calcium-chelate, and experiments on animals prove that the peptide calcium-chelate can significantly improve the maximum load and structural strength of the tibia of an osteoporosis mouse, and has a good effect of improving the bone biomechanical properties of an osteoporosis mouse.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of processing of bioactive products, and particularly relates to a Chlorella pyrenoidosa peptide calcium chelate and a preparation method thereof. BACKGROUND

[0002] Calcium is one of the most common inorganic elements in the human body, and is an essential nutrient for the human body, playing a key role in biological activities such as metabolism, muscle contraction, and signal transmission. With age, the phenomenon of bone calcium loss is increasingly 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, high blood pressure, kidney stones, etc. Although calcium supplements such as inorganic calcium, organic calcium and amino acid calcium provide sufficient calcium content, they ignore the absorption effect of calcium in the human body.

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

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

[0005] The present application provides a Chlorella pyrenoidosa calcium chelating peptide and a preparation method thereof, and a Chlorella pyrenoidosa peptide calcium chelate and a preparation method thereof. The prepared Chlorella pyrenoidosa peptide calcium chelate can enhance the maximum load and structural strength of the tibia of osteoporotic mice.

[0006] The present application first provides a Chlorella pyrenoidosa calcium-chelated peptide, and a preparation method thereof is as follows:

[0007] 1) Chlorella pyrenoidosa broken wall

[0008] The Chlorella pyrenoidosa and water are fully mixed, and the broken wall operation of repeated freeze-thawing, high temperature and high pressure, and high pressure homogenization is carried out respectively;

[0009] The fully mixed is that the Chlorella pyrenoidosa and water are mixed at a ratio of 1:10 (w / v), and a disperser is used to beat for 10 min, and the Chlorella pyrenoidosa and water are fully mixed; as preferred, repeated freeze-thawing is selected as the broken wall method, and the repeated freeze-thawing is that the Chlorella pyrenoidosa is frozen 3-4 times at-20 DEG C for 10-12 h each time, and thawed 3-4 times at room temperature.

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

[0011] The Chlorella pyrenoidosa solution after repeated freeze-thawing is subjected to high temperature and high pressure treatment; the high temperature and high pressure treatment is 121 DEG C, 0.1 MPa, and the treatment time is 15-20 min.

[0012] 3) Preparation of Chlorella pyrenoidosa calcium-chelated peptide

[0013] Protease is added for enzymolysis, and after the enzymolysis is completed, enzyme is inactivated, and the Chlorella pyrenoidosa calcium-chelated peptide is obtained by centrifugation and concentration and drying of the enzymolysis solution;

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

[0015] As preferred, the protease is alkaline protease and flavor protease;

[0016] The enzymolysis is carried out under the conditions of pH 7-9 and 50-55 DEG C for 4-6 h;

[0017] As preferred, the enzymolysis is carried out by adding 3.5-4% alkaline protease and flavor protease with a mass ratio of 3:1 (w / w) under the conditions of pH 8 and 50 DEG C water bath for 6 h.

[0018] The Chlorella pyrenoidosa calcium-chelated peptide has an amino acid sequence of DLEPVPGEEN (SEQ ID NO:1), DIEPVPGEENQY (SEQ ID NO:2), or DIEPVPGEE (SEQ ID NO:3).

[0019] The present application provides a Chlorella pyrenoidosa peptide calcium-chelated compound, and a preparation method thereof is as follows:

[0020] The chelated peptide of Chlorella pyrenoidosa is mixed with water to prepare a chelated peptide solution of Chlorella pyrenoidosa, and then calcium chloride dihydrate is added and mixed, and the chelation reaction is carried out under water bath condition, and after the reaction is completed, anhydrous ethanol is added, and the chelated peptide of Chlorella pyrenoidosa is obtained by centrifugation.

[0021] The chelated peptide solution of Chlorella pyrenoidosa is prepared by mixing the chelated peptide of Chlorella pyrenoidosa obtained by concentration and drying with water, and the mass concentration is 50-60 g / L.

[0022] The solution system of the chelation reaction is a mixed solution system of the chelated peptide solution of Chlorella pyrenoidosa and calcium chloride dihydrate, and the mass ratio of the chelated peptide of Chlorella pyrenoidosa to calcium ions is 5:1 (w / w).

[0023] Preferably, the chelation reaction is carried out under the condition of pH 9.5-10.0 and water bath at 25-30℃ for 20-30 min.

[0024] The chelated peptide of Chlorella pyrenoidosa is mixed with water to prepare a chelated peptide solution of Chlorella pyrenoidosa, and then calcium chloride dihydrate is added and mixed, and the chelation reaction is carried out under water bath condition, and after the reaction is completed, anhydrous ethanol is added, and the chelated peptide of Chlorella pyrenoidosa is obtained by centrifugation.

[0025] The chelated peptide of Chlorella pyrenoidosa can be used for preparing a calcium supplement preparation.

[0026] The chelated peptide of Chlorella pyrenoidosa can be used for preparing a calcium supplement preparation. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The figure is the calcium chelation activity and polypeptide yield of the alkaline protease enzyme hydrolysate of Chlorella pyrenoidosa treated by different wall breaking methods.

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

[0029] Figure 3 The figure is the calcium chelation activity and degree of hydrolysis of the enzyme hydrolysate of Chlorella pyrenoidosa under different complex enzyme ratios, enzyme hydrolysis times and enzyme amounts.

[0030] Figure 4 The figure is the calcium chelation condition optimization of Chlorella pyrenoidosa.

[0031] Figure 5 The figure is the effect of the chelated peptide of Chlorella pyrenoidosa on the bone biomechanics of osteoporotic mice. DETAILED DESCRIPTION

[0032] The present application uses the method of combining repeated freeze-thawing and high-temperature high-pressure treatment to pretreat Chlorella pyrenoidosa for enzymolysis. Compared with the enzymolysis product obtained before pretreatment, the enzymolysis product obtained after pretreatment has higher calcium chelating activity. By optimizing the conditions such as chelating temperature, chelating time, pH, and peptide calcium ratio, a high-calcium Chlorella pyrenoidosa peptide calcium chelate is prepared. Animal experiments show that the high-dose group of Chlorella pyrenoidosa peptide calcium chelate can significantly improve the tibial maximum load and structural strength of osteoporotic mice.

[0033] The present application will be described in detail below in combination with specific examples and drawings.

[0034] Example 1: Preparation of Chlorella pyrenoidosa peptide

[0035] The preparation of Chlorella pyrenoidosa peptide with high calcium chelating activity includes the following steps:

[0036] 1) Chlorella pyrenoidosa and water are mixed at a ratio of 1:10 (w / v) using a disperser to stir for 10 min, fully mixed, frozen 4 times in a-20℃ refrigerator, each time for 12 h, thawed at room temperature 4 times, and the repeatedly freeze-thawed Chlorella pyrenoidosa solution is adjusted to pH 6.5 with 0.1M NaOH, and then subjected to high-temperature high-pressure treatment at 121℃ and 0.1MPa for 15 min.

[0037] 2) Alkaline protease and flavor protease are added at a mass ratio of 3:1 (w / w), and the enzyme amount is 4%, the system pH is adjusted to 8.0, and the hydrolysis is carried out at 50℃ for 6 h for enzymolysis. After the enzymolysis is completed, the enzyme is inactivated by boiling, and the Chlorella pyrenoidosa calcium chelating peptide is obtained by centrifugation and drying of the enzymolysis solution;

[0038] In this example, Chlorella pyrenoidosa is first subjected to repeated freeze-thawing, high-temperature high-pressure, and high-pressure homogenization for cell wall disruption, and then subjected to enzymolysis with 4% alkaline protease to obtain enzymolysis products. As shown in Table 1, the polypeptide yield of the alkaline protease enzymolysis product of Chlorella pyrenoidosa treated by repeated freeze-thawing is lower than that of the enzymolysis products treated by the other two methods, but its calcium chelating activity is significantly higher than that of the enzymolysis products treated by the other two methods, indicating that repeated freeze-thawing can produce more peptides with calcium chelating activity. Figure 1 Figure 2 After the Chlorella pyrenoidosa subjected to repeated freeze-thawing is subjected to high-temperature high-pressure treatment, four enzymes, alkaline protease, neutral protease, papain, and flavor protease, are selected for enzymolysis to obtain enzymolysis products. As shown in Table 2, after high-temperature high-pressure treatment, the nitrogen recovery rate of the remaining three hydrolysis products is significantly improved, the degree of hydrolysis of the four hydrolysis products is decreased, the calcium chelating activity of the flavor protease hydrolysis product is significantly improved, and the proportion of the part with a molecular weight of less than 500 Da in the Chlorella pyrenoidosa enzymolysis product prepared by high-temperature high-pressure treatment is relatively high. ​

[0039] The alkaline protease and the flavor protease with high calcium chelating activity were selected for complex enzymolysis. The effects of different complex enzyme ratios, enzymolysis time and enzyme dosage on the calcium chelating activity were investigated by single factor experiment, and the results are shown in Figure 3 The calcium chelating activity of the enzymolysis product was high when the mass ratio of the flavor protease to the alkaline protease was 1:3 (w / w), the enzymolysis time was 6 h, and the enzyme dosage was 5%. According to the results of the single factor experiment, a three-factor and three-level orthogonal experiment was performed to optimize the enzymolysis process. As shown in Table 1, the optimal enzymolysis process for the protein Chlorella pyrenoidosa peptide with high calcium chelating activity was that the mass ratio of the flavor protease to the alkaline protease was 1:3 (w / w), the enzymolysis time was 6 h, and the enzyme dosage was 4%. After verification, the calcium binding capacity of the protein Chlorella pyrenoidosa peptide prepared in this embodiment was 13.2039 ± 1.6304 μg / mg.

[0040] Table 1: Orthogonal experiment table of enzymolysis process

[0041]

[0042] The peptide fingerprint of the protein Chlorella pyrenoidosa calcium chelating peptide was determined by high-resolution mass spectrometry. The protein database of Chlorella pyrenoidosa was added, and Proteome Discoverer was used for library searching. A total of 560 peptides were matched. It has been shown in previous studies that acidic amino acids have better calcium binding capacity due to containing two carboxyl groups. Therefore, the peptide sequences containing two or more acidic amino acids were screened out, as shown in Table 2.

[0043] Table 2: Sequence table of protein Chlorella pyrenoidosa calcium chelating peptide

[0044]

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

[0046] Example 2: Preparation of protein Chlorella pyrenoidosa peptide calcium chelate

[0047] The protein Chlorella pyrenoidosa calcium chelating peptide was added to water to make its mass concentration 50 g / L, then calcium ions with a peptide calcium mass ratio of 5:1 (w / w) were added, and stirred uniformly. Under the conditions of pH 10 and temperature 30°C, the mixture was water-bathed for 30 min. Eight times the volume of anhydrous ethanol was added, stirred uniformly and left to stand for 12 h. The precipitate was obtained by centrifugation, and the protein Chlorella pyrenoidosa peptide calcium chelate was obtained by drying.

[0048] In this embodiment, the protein Chlorella pyrenoidosa peptide calcium chelating conditions were optimized, and the results are shown in Figure 4As shown, four factors—chelation temperature, chelation time, peptide-calcium mass ratio, and pH—were selected for single-factor experiments. The optimal conditions for chelating Chlorella peptide-calcium using this process were determined to be a peptide-calcium mass ratio of 5:1 (w / w), pH 10, chelation temperature of 30℃, and chelation time of 30 min. Verification showed that the calcium content of the Chlorella peptide-calcium chelate prepared by this process was 192.0038 ± 7.9082 μg / mg.

[0049] Example 3: Detection of the effect of protein-nucleated Chlorella peptide-calcium chelate on improving bone biomechanical properties

[0050] This invention establishes an ovariectomized osteoporosis mouse model and administers a protein-nucleated Chlorella peptide calcium chelate to the mice via gavage. The specific steps are as follows:

[0051] 1. Animal husbandry and experimental design

[0052] Female C57BL / 6J mice were randomly divided into a sham-operated group and an ovariectomized group. In the ovariectomized group, both ovaries were removed, while in the sham-operated group, only a small amount of adipose tissue was removed after opening the abdominal cavity. Samples were administered via gavage starting one week post-operation and continued for 12 weeks. The specific groupings were as follows: ① Sham-operated group (SHAM, n=6): administered pure water via gavage at a dose of 0.2 mL per 20g of mouse body weight daily; ② Model group (MC, n=6): administered pure water via gavage at a dose of 0.2 mL per 20g of mouse body weight daily; ③ Positive control group (ALN, n=6): administered alendronate sodium via gavage at a dose of 1 mg / kg; ④ Calcium carbonate group (CaCO3, n=6): administered calcium carbonate (CaCO3) via gavage. 2+ The dosage was 104 mg / kg / d; ⑤ Low-dose Chlorella peptide calcium chelate group (CCL, n=6): Chlorella peptide calcium chelate (Ca) was administered by gavage. 2+ The dosage was 52 mg / kg / d; ⑥ High-dose Chlorella peptide calcium chelate group (CCH, n=6): Chlorella peptide calcium chelate (Ca) was administered by gavage. 2+ The dosage is 104 mg / kg / day.

[0053] 2. Animal-based materials

[0054] After 12 weeks of gavage, mice were harvested. Prior to harvesting, the mice were fasted for 12 hours. Blood was collected from the eyeballs, and the mice were euthanized by cervical dislocation. The left and right tibias were harvested and fixed in 4% neutral formaldehyde fixative. Bone biomechanics of the mouse tibias were measured using a small animal bone strength tester. Figure 5 As shown, the maximum load and structural strength of the MC group were significantly lower than those of the SHAM group, indicating that the osteoporosis model was successfully established 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 Chlorella pyrenoidosa peptide calcium chelate prepared by the application can improve the bone biomechanical properties of osteoporotic mice and reduce the risk of bone fracture.

Claims

1. A protein-nucleated Chlorella calcium chelate peptide, characterized in that, The amino acid sequence of the protein nucleus Chlorella calcium chelate peptide is DLEPVPGEEN, DIEPVPGEENQY, or DIEPVPGEE.

2. A protein-nucleated Chlorella peptide-calcium chelate, characterized in that, The preparation method of the protein-nucleus Chlorella peptide calcium chelate is as follows: The protein-nucleated Chlorella calcium chelate peptide described in claim 1 was mixed with water to prepare a protein-nucleated Chlorella calcium chelate peptide solution. Then, calcium chloride dihydrate was added, mixed, and a chelation reaction was carried out under water bath conditions. After the reaction was completed, anhydrous ethanol was added, and the mixture was centrifuged to obtain the protein-nucleated Chlorella peptide calcium chelate.

3. The protein-nucleus Chlorella peptide calcium chelate as described in claim 2, characterized in that, The protein-nucleated Chlorella calcium chelate peptide solution has a mass concentration of 50-60 g / L; the mass ratio of the protein-nucleated Chlorella calcium chelate peptide to calcium ions is 5:

1.

4. The protein-nucleus Chlorella peptide calcium chelate as described in claim 3, characterized in that, The chelation reaction was carried out under water bath conditions of pH 9.5-10.0 and 25-30 ℃ for 20-30 min.

5. The application of the protein nucleus Chlorella peptide calcium chelate according to claim 2 in the preparation of calcium supplements.

6. A calcium supplement, characterized in that, The calcium supplement contains a pharmacologically effective concentration of the protein-nucleated Chlorella peptide calcium chelate of claim 5.

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