An iron chelate of a phosphorylated peptide of hemoglobin of yanan yellow cattle, and a preparation method and application thereof

Abstract

This invention relates to the field of biomedical technology, providing a phosphorylated peptide iron chelate of Yanbian cattle hemoglobin, its preparation method, and its applications. The Yanbian cattle hemoglobin phosphorylated peptide iron chelate is prepared from Yanbian cattle blood through enzymatic hydrolysis, phosphorylation, and iron chelation. Specifically, the phosphorylation step actively introduces strongly negatively charged phosphate groups into the Yanbian cattle hemoglobin peptide chain, serving as new and stronger metal ion coordination sites, thereby specifically enhancing its chelating ability for ferrous ions and the stability of the complex. The Yanbian cattle hemoglobin phosphorylated peptide iron chelate possesses high iron content and good gastrointestinal stability, showing broad application prospects in the preparation of a new generation of highly efficient and low-irritant iron supplements.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a phosphorylated peptide iron chelate of Yanbian cattle hemoglobin, its preparation method, and its application. Background Technology

[0002] Iron is an essential trace element for the human body, directly involved in hemoglobin synthesis and oxygen transport. Iron deficiency anemia, a global health problem, is particularly common in children, women of childbearing age, and the elderly. Currently, the most commonly used oral iron supplements in clinical practice are still inorganic iron salts such as ferrous sulfate. Although these compounds are inexpensive, they have two major drawbacks: firstly, their absorption rate is low and easily interfered with by factors such as phytic acid and tannins in food; secondly, free iron ions significantly irritate the gastrointestinal mucosa, often causing side effects such as nausea and abdominal discomfort, leading to low medication adherence. Peptide iron chelates are considered a promising third-generation iron supplement. Their mechanism involves using small peptide fragments obtained from enzymatic protein hydrolysis, which form a stable cyclic coordination structure with ferrous ions through their terminal amino and carboxyl groups and side chain amino acid residues. These peptide iron complexes can protect iron ions to a certain extent, reduce gastrointestinal irritation, and may improve absorption efficiency through intestinal peptide transport channels.

[0003] Animal blood, especially bovine blood, is an important raw material choice in this field due to its high hemoglobin content and concentrated resources. my country produces a large amount of bovine blood annually from slaughter, but its effective utilization rate is low, with most being used only for primary processing or discarded. Developing deep-processed bovine blood protein products with high bioavailability and added value is of practical significance. Yanbian Yellow Cattle, a superior local beef cattle breed in my country, has excellent meat quality, and it is speculated that its hemoglobin may also have a unique amino acid composition or sequence. However, there are currently no in-depth studies or industrial reports on the systematic development of its hemoglobin into a highly effective iron supplement. Early studies have confirmed that the hemoglobin of Yanbian Yellow Cattle exhibits genetic diversity, and its blood protein genetic background differs significantly from other breeds. Shen Xiuji et al. tested the hemoglobin types of 114 Yanbian Yellow Cattle and found that the hemoglobin of Yanbian Yellow Cattle is governed by two genes, A and B, and exhibits three phenotypes: AA, AB, and BB. This indicates that there are indeed detectable inter-individual differences in the hemoglobin of this breed, i.e., genetic variation. (Reference: Shen Xiuji, Shen Xiangjun, Li Wenyong, et al. Preliminary study on the relationship between hemoglobin type (Hb) and production performance of Yanbian Yellow Cattle [J]. Journal of Yanbian Agricultural College, 1989, (01): 31-35.). Wang Yuying et al.'s study on the blood protein polymorphism of seven Chinese yellow cattle breeds found that, through analysis of multiple loci including hemoglobin, Yanbian Yellow Cattle are genetically distant from other breeds and form their own group in cluster analysis. This proves from the perspective of population genetics that the overall blood protein gene composition of Yanbian Yellow Cattle has its uniqueness. (Reference: Wang Yuying, Pang Zhihong, Cao Honghe, et al. Study on the blood protein polymorphism and its genetic relationship of some Chinese yellow cattle [J]. Journal of Animal Husbandry and Veterinary Medicine, 1991, (04): 296-301.) Despite the significant advantages of peptide-iron chelates, unmodified heme protein-digested peptides still face two major technical bottlenecks when chelating with iron. First, the iron-binding capacity is limited. The number and affinity of iron-binding sites in natural peptides are inherently limited by their amino acid sequences, making it difficult for the iron content of the final product to exceed a certain threshold, thus limiting the iron supplementation efficiency. Second, chelation stability needs improvement. In the complex gastrointestinal environment, especially facing low-pH gastric juice and the action of various proteases, some chelate bonds may break, leading to premature iron release. This not only reduces absorption efficiency but may also re-induce gastrointestinal irritation. Therefore, appropriate modification of the peptide chain to enhance its metal-chelating ability has become a key research direction.

[0004] Phosphorylation modification is a mature and safe technique in the field of food protein modification. It involves introducing a phosphate group (-PO3) onto the serine, threonine hydroxyl group, or lysine amino group of the peptide chain. 2-This introduces a large number of negatively charged sites into the molecule. Phosphate groups have a strong affinity for divalent and trivalent metal ions. Studies have reported that phosphorylation modification can significantly enhance the calcium-binding capacity of soybean protein or casein phosphopeptides.

[0005] Therefore, whether phosphorylation technology can be applied to bovine hemoglobin peptides to specifically enhance their chelating ability for ferrous ions and the stability of the complex has become an urgent issue to be explored in this field. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to overcome these shortcomings by providing a phosphorylated peptide iron chelate of Yanbian cattle blood hemoglobin, its preparation method, and its application, using Yanbian cattle blood hemoglobin as raw material. This method is simple, has mild conditions, and produces a product with high iron content and stable chelation.

[0007] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing a phosphorylated peptide iron chelate of hemoglobin from Yanbian cattle, the preparation method comprising the following steps: (1) Using Yanbian cattle blood as the raw material, bovine hemoglobin peptides were obtained by enzymatic hydrolysis after obtaining hemoglobin solution; (2) The bovine hemoglobin peptide is then mixed with a phosphorylation reagent and phosphorylated under alkaline conditions to obtain a phosphorylated hemoglobin peptide solution. (3) Add soluble ferrous salt to the phosphorylated hemoglobin peptide solution to conduct an iron chelation reaction and obtain the iron chelate of phosphorylated hemoglobin peptide of Yanbian cattle.

[0008] This invention demonstrates that applying phosphorylation technology to bovine hemoglobin peptides can specifically enhance the chelating ability of bovine hemoglobin peptides to ferrous ions and the stability of the complex. It also provides a process route for preparing peptide-iron chelates using Yanbian yellow bovine hemoglobin as raw material through enzymatic hydrolysis, phosphorylation, and chelation steps.

[0009] In this invention, the preparation method uses Yanbian Yellow Cattle blood as raw material. Yanbian Yellow Cattle, as a superior breed, may have hemoglobin with a unique amino acid sequence or conformation. Simultaneously, phosphorylation modification is a key improvement step, actively introducing strongly negatively charged phosphate groups (-PO3) onto the hemoglobin peptide chain of Yanbian Yellow Cattle through chemical modification. 2- It serves as a new and stronger metal ion coordination site; the resulting phosphorylated hemoglobin peptide has a high chelation rate with soluble ferrous salt, is stable in the gastrointestinal environment, can be effectively absorbed through peptide channels, has high bioavailability, and avoids the direct stimulation of the gastrointestinal tract by free iron, with few side effects.

[0010] As a preferred embodiment of the present invention, the phosphorylation reagent includes any one or a combination of at least two of sodium tripolyphosphate, sodium pyrophosphate, and phosphorus oxychloride.

[0011] Preferably, the soluble ferrous salt includes any one or a combination of at least two of ferrous chloride, ferrous sulfate, and ferrous acetate.

[0012] As a preferred technical solution of the present invention, the enzyme used in step (1) for enzymatic hydrolysis is papain.

[0013] Preferably, the papain has an enzyme activity of 700,000 U / g to 800,000 U / g; for example, it can be 700,000 U / g, 720,000 U / g, 740,000 U / g, 750,000 U / g, 760,000 U / g, 780,000 U / g, or 800,000 U / g, etc.

[0014] Preferably, the enzymatic hydrolysis temperature in step (1) is 35-45℃, for example, it can be 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃ or 45℃, etc.; the enzymatic hydrolysis time is 3-5 h, for example, it can be 3 h, 3.1 h, 3.2 h, 3.5 h, 3.6 h, 3.8 h, 4 h, 4.2 h, 4.4 h, 4.5 h, 4.6 h, 4.7 h, 4.8 h or 5 h, etc.

[0015] As a preferred technical solution of the present invention, the operation of mixing bovine hemoglobin peptide with phosphorylation reagent in step (2) is as follows: the bovine hemoglobin peptide is prepared into a bovine hemoglobin peptide solution of 8-12 mg / ml (for example, it can be 8 mg / ml, 8.5 mg / ml, 9 mg / ml, 9.5 mg / ml, 10 mg / ml, 10.5 mg / ml, 11 mg / ml, 11.5 mg / ml or 12 mg / ml, etc.), and then a phosphorylation reagent is added. The mass percentage of phosphorylation reagent in the resulting solution is 2%-10% (for example, it can be 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, etc., preferably 8%).

[0016] Preferably, the reaction temperature of the phosphorylation reaction in step (2) is 30-70℃, for example, it can be 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃ or 70℃, etc.; the reaction time is 30-150 min, for example, it can be 30 min, 35 min, 40 min, 50 min, 60 min, 80 min, 90 min, 100 min, 110 min, 120 min, 130 min, 140 min or 150 min, etc.

[0017] Preferably, the pH value of the alkaline conditions in step (2) is 7.0-10.0, for example, it can be 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 or 10.0.

[0018] As a preferred technical solution of the present invention, the mass ratio of phosphorylated hemoglobin peptide to soluble ferrous salt in step (3) is 1:2-4:1; for example, it can be 0.5:1, 0.8:1, 1:1, 1.2:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1 or 4:1, etc., preferably 3:1.

[0019] Preferably, the pH value of the iron chelation reaction in step (3) is 7.0-9.0, for example, it can be 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 or 10.0.

[0020] Preferably, the reaction temperature of the iron chelation reaction in step (3) is 30-70℃, for example, it can be 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃ or 70℃, etc.; the reaction time is 15-75 min, for example, it can be 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 65 min, 70 min or 75 min, etc.

[0021] As a preferred technical solution of the present invention, the preparation method includes the following steps: (1) Extraction and enzymatic hydrolysis of hemoglobin: Fresh Yanbian cattle blood was taken, anticoagulated, centrifuged, and subjected to freeze-thaw combined with ultrasonic disruption to obtain a pure hemoglobin solution. The pH and temperature of the hemoglobin solution were adjusted, and papain was added for enzymatic hydrolysis. After enzymatic hydrolysis, the enzyme was inactivated and decolorized. The supernatant was collected after centrifugation to obtain the hemoglobin hydrolysate. The hydrolyzed bovine hemoglobin peptides were ultrafiltered using ultrafiltration centrifuge tubes and freeze-dried to obtain bovine hemoglobin peptide powder.

[0022] (2) Phosphorylation modification: The above hemoglobin protein hydrolysate is mixed with a phosphorylation reagent (e.g., sodium tripolyphosphate STP), the pH of the reaction system is adjusted to alkaline, and the phosphorylation reaction is carried out at a certain temperature. After the reaction is completed, the solution is dialyzed to remove unreacted phosphates and small molecule impurities to obtain a phosphorylated hemoglobin peptide solution.

[0023] (3) Iron chelation: Add a soluble ferrous salt (e.g., ferrous chloride) to the phosphorylated hemoglobin peptide solution, adjust the pH to weakly alkaline, and react in the dark. After the reaction is complete, the solution is precipitated with alcohol, the precipitate is collected by centrifugation, and then freeze-dried to obtain the iron chelate powder of the phosphorylated hemoglobin peptide from Yanbian cattle.

[0024] For example, the preparation method described in this invention can be carried out according to the following steps: (1) Using Yanbian cattle blood as raw material, the blood was subjected to anticoagulation, centrifugation, freeze-thaw and ultrasonic disruption to obtain hemoglobin solution. The pH was adjusted to 5.5-6.5, and then papain was added for enzymatic hydrolysis. During the ultrasonic disruption process, the blood cell to water ratio was 1:(3-5), the interval ratio was 2:1, the ultrasonic power was 150 W, and the working time was 15-20 min. The papain has an enzyme activity of 700,000-800,000 U / g, a temperature of 35-45℃, a material-to-liquid ratio of 40-50 g / L, and an enzymatic hydrolysis time of 3-5 h. After the enzymatic hydrolysis is completed, the enzyme is inactivated and decolorized. After centrifugation, the supernatant is collected, and then the supernatant is subjected to ultrafiltration and freeze-drying to obtain bovine hemoglobin peptides. (2) Prepare a bovine hemoglobin peptide solution of 10 mg / ml, and then add a phosphorylation reagent. The mass percentage of the phosphorylation reagent in the resulting solution is 8%. The phosphorylation reaction is carried out at pH 7.0-10.0, the reaction temperature is 30-70℃, and the reaction time is 30-150 min to obtain a phosphorylated hemoglobin peptide solution. (3) Add ferrous chloride to the phosphorylated hemoglobin peptide solution, wherein the mass ratio of the phosphorylated hemoglobin peptide to the ferrous ions in the ferrous chloride is 1:2-4:1, and an iron chelation reaction is carried out at pH 7.0-9.0 and 30-70℃ for 15-75 min to obtain the iron chelate of the phosphorylated hemoglobin peptide of Yanbian cattle.

[0025] Secondly, the iron chelate of phosphorylated peptides of Yanbian cattle hemoglobin was prepared using the preparation method described in the first aspect.

[0026] This invention utilizes the abundant blood resources of Yanbian cattle to prepare a novel peptide-iron chelate through enzymatic hydrolysis, phosphorylation modification, and efficient chelation with ferrous ions. The peptide-iron chelate has a high iron content and good gastrointestinal stability. Its effect on improving iron deficiency anemia has been verified in animal models, thus providing a feasible technical solution for developing a new generation of efficient and low-irritant iron supplements.

[0027] Thirdly, the present invention provides the application of the Yanbian cattle hemoglobin phosphorylated peptide iron chelate as described in the second aspect in the preparation of iron supplements.

[0028] As a preferred embodiment of the present invention, the iron supplement is an oral preparation for the prevention or treatment of iron deficiency anemia.

[0029] Preferably, the iron supplement is a food or a medicine.

[0030] For example, the phosphorylated peptide iron chelate of the present invention can be used to prepare drugs or health foods for improving or treating iron deficiency anemia in mammals.

[0031] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The preparation method described in this invention applies phosphorylation technology to bovine hemoglobin peptides, providing a process route for preparing peptide iron chelates by using Yanbian yellow bovine hemoglobin as raw material through enzymatic hydrolysis, phosphorylation and chelation steps, and the yield of the obtained product can reach 60%.

[0033] (2) The phosphorylated peptide iron chelate prepared from Yanbian cattle hemoglobin is stable in the gastrointestinal environment, can be effectively absorbed through peptide channels, has high bioavailability, and avoids direct stimulation of the gastrointestinal tract by free iron, resulting in fewer side effects. Compared with traditional chemically synthesized iron supplements, it has better biocompatibility. Animal experiments show that it is significantly more effective than ordinary ferrous sulfate and unphosphorylated peptide iron chelate in improving hemoglobin, red blood cell count, and serum iron levels in rats with iron deficiency anemia. In addition, some peptide iron products use common protein sources, failing to fully utilize unique resources, resulting in low product differentiation and added value.

[0034] (3) This preparation method applies the high-value utilization of Yanbian cattle blood resources with regional characteristics to the development of iron supplements. It not only realizes the high-value utilization of slaughter by-products and turns waste into treasure, which is in line with the concept of green and sustainable development, but also gives the product a natural and distinctive raw material endorsement, thereby enhancing the product's potential market value and competitiveness. Attached Figure Description

[0035] Figure 1 The image shows a comparison of the infrared spectra of the phosphorylated peptide iron chelate prepared in Example 1 and the unphosphorylated peptide iron chelate prepared in Comparative Example 1.

[0036] Figure 2 Figure 1 shows the results of ferrous ion chelating ability, phosphorylation degree, chelation rate, and chelate yield of the products under different conditions in Example 1. Figure I shows the results of different STP concentrations (different uppercase letters A, B, and C indicate significant differences in phosphorylation degree, and different lowercase letters a, b, and c indicate Fe). 2+ The differences in chelation capacity were significant (p < 0.05). Figure II shows the peptide-iron mass ratio (different uppercase letters A, B, C, D, E indicate significant differences in chelation rate, and different lowercase letters a, b, c, d, e indicate significant differences in chelate yield (p < 0.05)).

[0037] Figure 3 This is a comparison chart of iron retention rates of different products during simulated gastric digestion in Test Example 2; the lowercase letters a, b, c, and d represent the effects of different iron supplements on the same substance at different digestion times.

[0038] Figure 4 This is a comparison chart of iron retention rates of different products during simulated intestinal digestion in Test Example 2; the lowercase letters a, b, and c represent the effects of different iron supplements on the same substance at different digestion times.

[0039] Figure 5 The graph shows the trend of hemoglobin concentration (Hb) over time in different intervention groups of iron-deficiency anemic rats during the 28-day experimental period in Test Example 3. Different lowercase letters indicate significant differences between groups (p < 0.05). Among them, Figure I is hemoglobin content, Figure II is red blood cell count, Figure III is hematocrit, Figure IV is mean corpuscular volume, Figure V is mean corpuscular hemoglobin content, and Figure VI is mean corpuscular hemoglobin concentration. Detailed Implementation

[0040] The technical solution of the present invention will be further illustrated below through specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be determined by the claims.

[0041] In the following examples, papain was purchased from Beijing Solarbio Science & Technology Co., Ltd. Unless otherwise specified, all other reagents and consumables used were purchased from conventional reagent manufacturers in the field; unless otherwise specified, all experimental methods and techniques used were conventional methods and techniques in the field.

[0042] In the following examples, the reagents used to adjust the pH were HCl and NaOH, both with a concentration of 1 mol / L.

[0043] In the following embodiments, the relevant abbreviations are as follows: Bovine hemoglobin peptide: BHP; Bovine hemoglobin peptide iron chelate: BHP-Fe; Phosphorylated bovine hemoglobin peptide iron chelate: P-BHP-Fe; Low-dose bovine hemoglobin peptide iron chelate: BHP-Fe-L; High-dose bovine hemoglobin peptide iron chelate: BHP-Fe-H; Low-dose phosphorylated bovine hemoglobin peptide iron chelate: P-BHP-Fe-L; High-dose phosphorylated bovine hemoglobin peptide iron chelate: P-BHP-Fe-H.

[0044] Example 1: Preparation of phosphorylated peptide iron chelate

[0045] This embodiment provides a method for preparing phosphorylated peptide iron chelates, the specific steps of which are as follows: (1) Preparation and enzymatic hydrolysis of hemoglobin solution Fresh Yanbian cattle blood was treated with 0.5% sodium citrate as an anticoagulant and stored at 4°C. Impurities were removed by filtration through three layers of gauze. The blood was centrifuged at 3500 rpm for 15 min to obtain the lower layer of red blood cells. An equal volume of 0.9% physiological saline was added for washing, followed by centrifugation at 3500 rpm for 10 min. The supernatant was discarded, and this process was repeated three times to obtain purified red blood cells. The fresh red blood cells were first frozen at -18°C for 12 h, thawed with running water, and then sonicated using an ultrasonic cell disruptor. The sonication was performed at a blood-to-water ratio of 1:4, an intermittent ratio of 2:1, an ultrasonic power of 150 W, and a working time of 20 min. The blood was then centrifuged at 4°C and 8000 rpm for 10 min, and the precipitate was discarded to obtain a Yanbian cattle hemoglobin solution.

[0046] Bovine hemoglobin solution was prepared with a solid-liquid ratio of 40 g / L. The pH was adjusted to 6.0, and the temperature was 40℃. Papain with an enzyme activity of 800,000 U / g was added, and the solution was hydrolyzed for 3 h. The enzyme was then inactivated in a 100℃ water bath for 10 min. After cooling, the solution was centrifuged at 8000 rpm for 10 min, and the supernatant was collected. The supernatant was decolorized using 2% activated carbon, shaken at 70℃ for 10 min, and allowed to stand for 1 h. The solution was then centrifuged at 8000 rpm for 10 min, and the supernatant was collected. The hydrolyzed bovine hemoglobin peptides were ultrafiltered using ultrafiltration centrifuge tubes with a molecular weight <1 kDa. The solution was freeze-dried to obtain BHP powder.

[0047] (2) Phosphorylation modification A hemoglobin peptide solution was prepared from the above-mentioned hemoglobin peptide powder with a molecular weight <1 kDa, wherein the peptide concentration was 10 mg / mL. Sodium tripolyphosphate (STP) was then added to achieve a STP mass percentage concentration of 8%. The pH was adjusted to 9.0 with NaOH solution, and the reaction was carried out in a 40°C water bath for 2 h. The reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 1 kDa and dialyzed against deionized water for 24 h to remove small molecule impurities, yielding a phosphorylated hemoglobin peptide solution. This solution was then freeze-dried to obtain P-BHP.

[0048] (3) Iron chelation The lyophilized P-BHP powder was diluted to 50 mg / mL with deionized water, and ferrous chloride was added to control the peptide to ferrous ion mass ratio at 3:1. The pH was adjusted to 7 with HCl solution, and the reaction was carried out at 50°C with stirring in the dark for 45 min. After the reaction was completed, 7 volumes of anhydrous ethanol were added, and the mixture was allowed to stand overnight at 4°C. The precipitate was collected by centrifugation at 10,000 rpm for 15 min, and then lyophilized to obtain a powdered product, namely P-BHP-Fe.

[0049] The yield of the product was determined to be 62 g / 100 g.

[0050] Example 2

[0051] The difference between this embodiment and Example 1 is that the STP concentration is adjusted to 2% in the phosphorylation modification step; the remaining steps are the same as in Example 1.

[0052] Example 3

[0053] The difference between this embodiment and Example 1 is that the STP concentration is adjusted to 4% in the phosphorylation modification step; the remaining steps are the same as in Example 1.

[0054] Example 4

[0055] The difference between this embodiment and Example 1 is that the STP concentration is adjusted to 6% in the phosphorylation modification step; the remaining steps are the same as in Example 1.

[0056] Example 5

[0057] The difference between this embodiment and Example 1 is that the STP concentration is adjusted to 10% in the phosphorylation modification step; the remaining steps are the same as in Example 1.

[0058] Example 6

[0059] The difference between this embodiment and Example 1 is that, in the iron chelation step, the mass ratio of peptide to ferrous ion is 1:2; the remaining steps are the same as in Example 1.

[0060] Example 7

[0061] The difference between this embodiment and Example 1 is that, in the iron chelation step, the mass ratio of peptide to ferrous ion is 1:1; the remaining steps are the same as in Example 1.

[0062] Example 8

[0063] The difference between this embodiment and Example 1 is that, in the iron chelation step, the mass ratio of peptide to ferrous ion is 2:1; the remaining steps are the same as in Example 1.

[0064] Example 9

[0065] The difference between this embodiment and Example 1 is that, in the iron chelation step, the mass ratio of peptide to ferrous ion is 4:1; the remaining steps are the same as in Example 1. Comparative Example 1: Preparation of Unphosphorylated Peptide Iron Chelate

[0066] Except for the absence of phosphorylation modification in step 3, the steps in this comparative example are exactly the same as those in Example 1. The heme protein hydrolysate is directly used for iron chelation reaction to obtain unphosphorylated peptide iron chelates.

[0067] (1) Preparation and enzymatic hydrolysis of hemoglobin solution Fresh Yanbian cattle blood was treated with 0.5% sodium citrate as an anticoagulant and stored at 4°C. Impurities were removed by filtration through three layers of gauze. The blood was centrifuged at 3500 rpm for 15 min to obtain the lower layer of red blood cells. An equal volume of 0.9% physiological saline was added for washing, followed by centrifugation at 3500 rpm for 10 min. The supernatant was discarded, and this process was repeated three times to obtain purified red blood cells. The fresh red blood cells were first frozen at -18°C for 12 h, thawed with running water, and then sonicated using an ultrasonic cell disruptor. The sonication was performed at a blood-to-water ratio of 1:4, an intermittent ratio of 2:1, an ultrasonic power of 150 W, and a working time of 20 min. The blood was then centrifuged at 4°C and 8000 rpm for 10 min, and the precipitate was discarded to obtain a bovine hemoglobin solution.

[0068] Bovine hemoglobin solution was prepared with a solid-liquid ratio of 40 g / L, pH adjusted to 6.0, and temperature maintained at 40℃. Papain with an enzyme activity of 800,000 U / g was added, and after enzymatic hydrolysis for 3 h, the enzyme was inactivated in a 100℃ water bath for 10 min. After cooling, the solution was centrifuged at 8000 rpm for 10 min, and the supernatant was collected. The supernatant was decolorized using 2% activated carbon, shaken at 70℃ for 10 min, and allowed to stand for 1 h. After centrifugation at 8000 rpm for 10 min, the supernatant was collected and freeze-dried to obtain bovine hemoglobin peptides. The hydrolyzed bovine hemoglobin peptides were ultrafiltered using ultrafiltration centrifuge tubes with a molecular weight <1 kDa, and then freeze-dried to obtain BHP powder.

[0069] (2) Iron chelation The lyophilized BHP powder was diluted to 50 mg / mL with deionized water, and ferrous chloride was added to control the peptide to ferrous ion mass ratio at 3:1. The pH was adjusted to 7 with HCl solution, and the reaction was carried out at 50°C with stirring in the dark for 45 min. After the reaction was completed, 7 volumes of anhydrous ethanol were added, and the mixture was allowed to stand overnight at 4°C. The mixture was centrifuged at 10,000 rpm for 15 min, and the precipitate was collected. After lyophilization, a brown powdery product, namely BHP-Fe, was obtained. Test Example 1: Fourier Transform Infrared Spectroscopy Analysis

[0070] The products obtained in Example 1 and Comparative Example 1 were subjected to Fourier transform infrared spectroscopy (FTIR).

[0071] A small amount of dried sample was mixed with KBr and extruded into a transparent sheet, which was then loaded onto an FTIR sample plate. Each sample was placed at 4000-4000 cm⁻¹. -1 The scan was performed within the wavenumber range, with a resolution of 2 cm⁻¹. -1 .

[0072] Figure 1 The results showed that BHP was at 3419.79 cm. -1 The absorption peak at that point is due to the stretching vibration of OH or NH. After phosphorylation modification, the wavenumber shifts to a lower value of 3348.71 cm⁻¹. -1 This is likely due to the phosphate group being linked to the oxygen atom on the hydroxyl group and the nitrogen atom on the amino group, confirming the successful introduction of the phosphate group.

[0073] After chelation with iron, P-BHP was at 3348.71 cm⁻¹. -1 The absorption peak at [location] shifted to 3294.42 cm⁻¹. -1 The presence of amino and carboxyl groups indicates that amino and carboxyl groups are involved in the formation of the peptide iron chelate.

[0074] P-BHP at 1543.05 cm -1 The absorption peak at that point shifts to a lower frequency (1540.76 cm⁻¹) after chelating iron ions. -1 This is due to the tensile vibration of C=O.

[0075] 980-920 cm⁻¹ in the infrared spectrum -1 The vibration in this range is due to the stretching vibration of P-OH, and the absorption peak of P-BHP is at 974.05 cm⁻¹. -1 At this location, after iron chelation, the frequency shifts to a lower frequency of 924.11 cm⁻¹. -1 .

[0076] In this invention, Example 1 is also compared with other examples, and the results are as follows: Figure 2 As shown: A comparison of Examples 1 and 2-5 (i.e., Figure I) shows that as the STP concentration increases within the range of 2% to 8%, the phosphorylation degree of bovine hemoglobin peptides and Fe... 2+The chelating abilities showed a significant upward trend, peaking at 8%. This is attributed to the increased effective collision probability in the system due to the higher concentration of the phosphorylating agent, thereby promoting the nucleophilic substitution reaction of the phosphate group on the hydroxyl group of the peptide side chain. When the concentration was further increased to 10%, both indicators declined, possibly because excess phosphate ions formed competitive binding in the system, hindering the effective coordination of iron ions with the active site of the peptide chain. Therefore, 8% STP was chosen for subsequent experiments.

[0077] A comparison of Examples 1 and 6-9 (i.e., Figure II) shows that when the peptide-iron mass ratio is 1:2 and 1:1, the chelation rate and chelate yield are significantly reduced. This indicates that at lower ratios, there is an excess of iron ions relative to the peptide coordination sites, resulting in a large number of iron ions not being effectively captured. However, when the ratio reaches 3:1, the active sites provided by the peptide, such as phosphate groups, carboxyl groups, and amino groups, achieve the optimal coordination state with iron ions. Further increasing the peptide-iron mass ratio (4:1) leads to a decrease in chelation rate and chelate yield. At high concentrations, peptide molecules may aggregate or form weak interactions such as intermolecular hydrogen bonds and van der Waals forces, altering the conformation of the peptide molecules and reducing the exposure of their binding sites. This results in a decrease in the number of free peptide segments that effectively bind iron ions, thus reducing the overall chelation rate and chelate yield. Test Example 2: In Vitro Static Gastrointestinal Digestion Simulation

[0078] To evaluate the stability of the product of the present invention in the digestive tract environment, an in vitro static gastrointestinal digestion model was used to simulate the digestion of the iron chelates obtained in Example 1 and Comparative Example 1, respectively, with ferrous chloride as a control.

[0079] 1. Samples: Phosphorylated peptide iron chelate P-BHP-Fe prepared in Example 1, unphosphorylated peptide iron chelate BHP-Fe prepared in Comparative Example 1, and ferrous chloride.

[0080] 2. Simulated gastric juice digestion of SGF: 10 mg / mL BHP-Fe and P-BHP-Fe solutions were mixed with 8 mL of SGF, and the pH was adjusted to 3.0. Subsequently, 0.134 g of pepsin and 5 μL of calcium chloride (0.3 mol / L) were added to the mixture, and the reaction was carried out at 37 °C for 2 h. Samples were taken at 0, 30, 60, 90, and 120 min, centrifuged at 10000 rpm for 10 min, and 1 mL of the sample solution was mixed with 1 mL of 1 mol / L HCl solution, 1 mL of 100 mg / mL hydroxylamine hydrochloride, 1 mL of 1.2 mg / mL ferroazine, and 5 mL of 100 mg / mL sodium acetate. The mixture was reacted at 20 °C for 30 min, and the absorbance was measured at 510 nm. The free iron content in the supernatant was determined.

[0081] 3. Simulated intestinal fluid digestion of SIF: After SGF digestion, the pH was adjusted to 7.0, and the mixture was further reacted with 16 mL of SIF, 0.2724 g of porcine bile salts, 0.1 g of trypsin, and 40 μL of calcium chloride (0.3 mol / L) at 37 °C for 2 h. Samples were taken at 0, 30, 60, 90, and 120 min, and the mixture was first inactivated at 90 °C for 15 min, followed by centrifugation at 10000 rpm for 10 min. 1 mL of the sample solution was mixed with 1 mL of 1 mol / L HCl solution, 1 mL of 100 mg / mL hydroxylamine hydrochloride, 1 mL of 1.2 mg / mL ferroazine, and 5 mL of 100 mg / mL sodium acetate. The mixture was reacted at 20 °C for 30 min, and the absorbance was measured at 510 nm.

[0082] 4. The iron retention rate is used as an indicator to evaluate stability.

[0083] The results are as follows Figure 3 (Simulated gastric juice) and Figure 4 As shown in the image (simulated intestinal fluid).

[0084] like Figure 3 As shown, under simulated gastric juice conditions, the iron retention rate of the three iron supplements decreased with the extension of gastric digestion time, indicating that iron is unstable in the gastric digestive environment such as gastric acid and is prone to chemical reactions with gastric acid to form insoluble precipitates or other unusable forms.

[0085] The iron retention rate of P-BHP-Fe is significantly better than that of BHP-Fe and FeCl2, attributed to the coordination enhancement and steric protection effect of the phosphate group. Phosphorylation modification may increase the negative charge of the molecule, enhancing its binding ability with iron, and may also reduce the approach of pepsin through steric hindrance, thereby delaying peptide chain degradation. In addition, the phosphate group itself may form a more stable coordination bond with iron, further reducing iron release and oxidation.

[0086] Therefore, the iron retention rate of P-BHP-Fe decreases more slowly than that of unmodified BHP-Fe.

[0087] like Figure 4 As shown, after a complete 4-hour simulated gastrointestinal digestion, the iron retention rate of P-BHP-Fe remained at around 90%, while that of BHP-Fe decreased to around 83%, and the iron retention rate of ferrous chloride was below 70%.

[0088] The results clearly show that phosphorylation modification greatly enhances the stability of peptide iron chelates in the gastrointestinal environment, which is beneficial for iron protection and targeted delivery. Test Example 3: Simulated Iron Deficiency Anemia in Rats

[0089] 1. Establishment of an iron deficiency anemia rat model: Healthy SD rats were selected and fed a low-iron diet and deionized water. After 28 days, the model was considered successful when the rat hemoglobin content was lower than 100 g / L.

[0090] 2. Grouping and drug administration: Rats that successfully developed the model were randomly divided into 7 groups: Model control group (Control); The ferrous sulfate group (2 mg Fe / kg BW) is designated as the FeSO4 group. The low-dose unphosphorylated peptide iron group (1 mg Fe / kg BW) (Comparative Example 1 product) is designated as the BHP-Fe-L group; The high-dose unphosphorylated peptide iron group (4 mg Fe / kg BW) (Comparative Example 1 product) is designated as the BHP-Fe-H group; The low-dose phosphorylated peptide iron group (1 mg Fe / kg BW) (product of Example 1) is designated as the P-BHP-Fe-L group; The high-dose phosphorylated peptide iron group (4 mg Fe / kg BW) (product of Example 1) is designated as the P-BHP-Fe-H group.

[0091] A separate normal control group (Model) was set up.

[0092] Each group was administered the drug by gavage according to the equivalent iron content for 28 days.

[0093] 3. Figure 5 The effects of different iron supplements on routine blood parameters in rats were demonstrated, including hemoglobin content (I), red blood cell count (II), hematocrit (III), mean corpuscular volume (IV), mean corpuscular hemoglobin content (V), and mean corpuscular hemoglobin concentration (VI).

[0094] The statistically significant differences (p < 0.05) indicated by different lowercase letters clearly demonstrate the effects between the treatment groups.

[0095] Compared with the blank control group, the model group showed significant reductions in all six red blood cell indicators, indicating that the rat model of iron deficiency anemia was successfully established.

[0096] The ferrous sulfate treatment group significantly improved various erythrocyte parameters in the model group. The BHP-Fe and P-BHP-Fe treatment groups showed better effects than the ferrous sulfate group in increasing hemoglobin content, erythrocyte count, and hematocrit.

[0097] Notably, the P-BHP-Fe group showed a more significant recovery in mean erythrocyte volume and mean erythrocyte hemoglobin content, suggesting potentially superior bioavailability or more stable release characteristics. This result indicates that phosphorylated BHP-Fe possesses a more durable or targeted mechanism of action in improving erythrocyte parameters.

[0098] The phosphorylated peptide iron chelate prepared in this invention showed the best effect in restoring hematological indicators in anemic rats, significantly better than ferrous sulfate and unphosphorylated peptide iron chelate, and approached normal levels, demonstrating its excellent anti-anemia activity and bioavailability.

[0099] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a phosphorylated peptide iron chelate of Yanbian cattle hemoglobin, characterized in that, The preparation method includes the following steps: (1) Using Yanbian cattle blood as the raw material, bovine hemoglobin peptides were obtained by enzymatic hydrolysis after obtaining hemoglobin solution; (2) The bovine hemoglobin peptide is then mixed with a phosphorylation reagent and phosphorylated under alkaline conditions to obtain a phosphorylated hemoglobin peptide solution. (3) Add soluble ferrous salt to the phosphorylated hemoglobin peptide solution to conduct an iron chelation reaction and obtain the iron chelate of phosphorylated hemoglobin peptide of Yanbian cattle.

2. The preparation method according to claim 1, characterized in that, The phosphorylation reagent includes any one or a combination of at least two of sodium tripolyphosphate, sodium pyrophosphate, or phosphorus oxychloride. The soluble ferrous salt includes any one or a combination of at least two of ferrous chloride, ferrous sulfate, or ferrous acetate.

3. The preparation method according to claim 1 or 2, characterized in that, The enzyme used in step (1) for enzymatic hydrolysis is papain; The papain has an enzyme activity of 700,000-800,000 U / g; The enzymatic hydrolysis temperature in step (1) is 40℃, and the enzymatic hydrolysis time is 3-5 h.

4. The preparation method according to any one of claims 1-3, characterized in that, The operation of mixing bovine hemoglobin peptide with phosphorylation reagent in step (2) is as follows: the bovine hemoglobin peptide is prepared into a bovine hemoglobin peptide solution of 8-12 mg / ml, and then phosphorylation reagent is added. The mass percentage of phosphorylation reagent in the resulting solution is 2%-10%. The phosphorylation reaction in step (2) is carried out at a temperature of 30-70°C for a time of 30-150 min. The pH value of the alkaline conditions described in step (2) is 7.0-10.

0.

5. The preparation method according to any one of claims 1-4, characterized in that, In step (3), the mass ratio of the phosphorylated hemoglobin peptide to the soluble ferrous salt is (0.5-4):

1. The pH value of the iron chelation reaction in step (3) is 7.0-9.0; The iron chelation reaction in step (3) is carried out at a temperature of 30-70℃ and for a time of 15-75 min.

6. The preparation method according to any one of claims 1-5, characterized in that, The preparation method includes the following steps: (1) Using Yanbian cattle blood as raw material, the blood was subjected to anticoagulation, centrifugation, freeze-thaw and ultrasonic disruption to obtain hemoglobin solution. The pH was adjusted to 5.5-6.5, and then papain was added for enzymatic hydrolysis. The papain has an enzyme activity of 700,000-800,000 U / g, a temperature of 35-45℃, and a hydrolysis time of 3-5 h. After the enzymatic hydrolysis is completed, the enzyme is inactivated and decolorized. After centrifugation, the supernatant is collected, and then the supernatant is subjected to ultrafiltration and freeze-drying to obtain bovine hemoglobin peptides. (2) Prepare a bovine hemoglobin peptide solution of 8-12 mg / ml, add a phosphorylation reagent, and the mass percentage of the phosphorylation reagent in the resulting solution is 2%-10%. The phosphorylation reaction is carried out at pH 7.0-10.0, the reaction temperature is 30-70℃, and the reaction time is 30-150 min to obtain a phosphorylated hemoglobin peptide solution. (3) Add ferrous chloride to the phosphorylated hemoglobin peptide solution, wherein the mass ratio of the phosphorylated hemoglobin peptide to the ferrous ions in the ferrous chloride is (0.5-4):1, and an iron chelation reaction is carried out at pH 7.0-9.0 and 30℃-70℃ for 15-75 min to obtain the iron chelate of the phosphorylated hemoglobin peptide of Yanbian cattle.

7. The iron chelate of phosphorylated peptide of Yanbian cattle hemoglobin prepared by the preparation method according to any one of claims 1-6.

8. The application of the Yanbian Yellow Cattle hemoglobin phosphorylated peptide iron chelate as described in claim 7 in the preparation of iron supplements.

9. The application according to claim 8, characterized in that, The iron supplement is an oral preparation for the prevention or treatment of iron deficiency anemia; The iron supplement is either a food or a medicine.

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