Yak whole blood polypeptide iron chelate as well as preparation method and application thereof

Abstract

The invention discloses a yak whole blood polypeptide iron chelate as well as a preparation method and application thereof, and relates to the technical field of medical materials. The method comprises the following steps: adding an anticoagulant into yak blood, and repeatedly freezing and thawing to obtain yak whole blood freeze-dried powder; performing enzymolysis on the freeze-dried powder in a water solvent to obtain a yak whole blood polypeptide solution; the preparation method comprises the following steps: carrying out ultrasonic treatment on a yak whole blood polypeptide solution, diluting, adding a ferrous chloride solution containing ascorbic acid, stirring to carry out chelation reaction, adjusting the pH value of the solution to be neutral, continuously stirring to carry out chelation reaction, and purifying the reactant to obtain the yak whole blood polypeptide iron chelate. The chelate prepared by the invention has the advantages of high iron loading rate, high stability and excellent dispersity, and is better in the aspects of intestinal solubility and biocompatibility.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and in particular to a yak whole blood polypeptide iron chelate, a preparation method and an application thereof. Background Art

[0002] Iron deficiency anemia is a common nutritional deficiency worldwide, affecting approximately 15% of the world's population. It significantly impacts the immune system, intellectual development, and physical strength, thus requiring extensive attention and timely intervention. Traditional iron supplements, such as ferrous sulfate and ferrous chloride, while widely used, suffer from poor solubility, low iron bioavailability, and intestinal irritation, which compromise the effectiveness of iron supplementation and reduce patient compliance.

[0003] Yaks live in high-altitude, low-oxygen environments. Studies have shown that hemoglobin (Hb) in yak blood has a higher oxygen affinity than regular bovine hemoglobin. This suggests that components in yak blood can more efficiently bind and transport iron ions, a property that has important application value in the development of iron supplements. Yak blood, a rich biological resource, is often considered a waste product and can contribute to environmental pollution. However, yak blood is rich in protein, and due to its natural origin and biocompatibility, its protein components have a lower risk of immune response and toxicity when used in iron supplements, making it an ideal raw material for the preparation of safe and effective iron supplements. Studies have reported methods for preparing iron supplements using yak blood as a raw material, and the prior art discloses a method for preparing cheese supplemented with yak blood meal, which can be used as an iron-supplementing food. However, the cheese made from the fermented and subsequently processed yak blood meal used has a relatively low iron content, representing only the iron content of the blood itself, resulting in a low iron loading rate. Therefore, there is a need to develop an iron-supplementing food with high iron loading rate, high solubility, high biocompatibility, and low intestinal irritation. Summary of the Invention

[0004] In view of the deficiencies in the above-mentioned background technology, the present invention mainly solves the deficiencies of the iron supplements in the prior art in terms of solubility, bioavailability and intestinal irritation side effects. The present invention provides a yak whole blood polypeptide iron chelate as well as a preparation method and application. The method first adds an anticoagulant to the whole blood of the yak, performs repeated freeze-thaw treatment to release the protein, and then obtains a lyophilized powder by freeze-drying; then uses alkaline protease Alcalase to enzymatically hydrolyze the blood to obtain yak whole blood polypeptides; then, ferrous chloride is mixed with ascorbic acid to increase the solubility, and finally a step-by-step chelation reaction is performed to achieve efficient binding of iron and polypeptides. The chelate prepared by the present invention has a high iron loading rate, high stability and excellent dispersibility. Compared with conventional iron supplements, it performs better in terms of intestinal solubility and biocompatibility. The chelate can not only improve the absorption efficiency of iron, but also effectively reduce the common side effects of traditional iron supplements, showing a wide range of potential applications in iron supplementation therapy.

[0005] The first object of the present invention is to provide a method for preparing a yak whole blood polypeptide iron chelate, comprising the following steps:

[0006] After adding anticoagulant to yak blood, the blood was repeatedly frozen and thawed to release protein, and then freeze-dried to obtain yak whole blood freeze-dried powder;

[0007] The lyophilized powder is enzymatically hydrolyzed in a water solvent to obtain a yak whole blood polypeptide solution;

[0008] Evenly dissolving ferrous chloride and ascorbic acid in a water solvent to obtain a ferrous chloride solution;

[0009] The yak whole blood polypeptide solution is ultrasonically treated and diluted, and then a ferrous chloride solution is added and stirred for a chelation reaction. The pH of the solution is then adjusted to neutral, and the stirring is continued for a chelation reaction. The reactant is then purified to obtain a yak whole blood polypeptide iron chelate.

[0010] Preferably, the volume ratio of the yak blood to the anticoagulant is 5 to 10:1;

[0011] Each freeze-thaw cycle involves freezing the yak blood at -30°C to -20°C for 12 to 36 hours, then thawing it in a 36°C to 38°C water bath until it is completely dissolved. The freeze-thaw cycle is repeated 3 to 5 times.

[0012] The freeze-drying temperature is -85 to -75°C and the time is 8 to 24 hours.

[0013] Preferably, the enzymatic hydrolysis process comprises: after the lyophilized powder is uniformly dispersed in an aqueous solvent, adding alkaline protease Alcalase for enzymatic hydrolysis; wherein the enzymatic hydrolysis time is 1 to 5 hours, the enzymatic hydrolysis temperature is 30 to 70° C.; the enzyme amount is 1 to 5% of the volume; and the mass concentration of the lyophilized powder uniformly dispersed in the aqueous solvent is 10 to 50 mg / mL.

[0014] Preferably, after the enzymatic hydrolysis is completed, the method further comprises: heating the enzymatic hydrolysis product in a 100° C. water bath for 10 to 20 minutes, rapidly cooling the product in an ice bath, centrifuging the solution, collecting the supernatant, and filtering the supernatant using a 0.22 μm filter to obtain a yak whole blood polypeptide solution.

[0015] Preferably, the mass ratio of ferrous chloride to ascorbic acid is 1 to 3:1, and after mixing, the mixture is sealed and placed at room temperature for 3 to 5 hours.

[0016] Preferably, when the ferrous chloride solution is added and stirred for the chelation reaction, the concentration of the polypeptide in the chelation reaction is 1 to 5 mg / mL; the mass ratio of ferrous chloride to polypeptide is 1:2 to 1:10; the reaction is stirred at 20 to 60° C., and the chelation reaction time is 60 to 120 minutes;

[0017] The ultrasonic treatment includes an ultrasonic amplitude of 20% to 40% and an ultrasonic time of 5 to 20 minutes.

[0018] Preferably, the pH of the solution is adjusted to neutral, and the chelation reaction is continued by stirring, which comprises: adjusting the pH to neutral with 0.5-1 M NaOH, and continuing the stirring reaction at 20-60° C. for 30-60 minutes.

[0019] Preferably, the purification process comprises:

[0020] The solution after the chelation reaction is stirred continuously and collected in a dialysis bag with a molecular weight cutoff of 100Da to 500Da. Deionized water is used as the dialysis medium and dialyzed at 2 to 6°C for 24 to 48 hours. The dialysate is replaced every 4 to 6 hours until the unbound iron ions and impurities are completely removed. After the dialysis is completed, the dialysate is frozen and transferred to a vacuum freeze dryer for freeze drying.

[0021] The second object of the present invention is to provide a yak whole blood polypeptide iron chelate.

[0022] The third object of the present invention is to provide a use of a yak whole blood polypeptide iron chelate in the preparation of iron-supplementing medicines or iron-supplementing health products.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention provides a yak whole blood polypeptide iron chelate, as well as a preparation method and application. This invention utilizes a staged reaction process to prepare a chelate with excellent dispersibility, high stability, high iron loading rate, excellent solubility, and bioavailability, while also reducing intestinal irritation. During the preparation process, strict control of enzymatic hydrolysis conditions, including enzyme dosage, temperature, and time, is combined with an innovative chelation reaction process to ensure the chelate's high iron loading rate and solubility. This invention effectively overcomes the shortcomings of traditional methods, such as low iron loading rate, poor gastrointestinal solubility of iron supplements, and intestinal irritation caused by free iron.

[0025] In order to ensure the reduced state and high solubility of iron ions during the reaction, the present invention uses ascorbic acid and ferrous chloride to dissolve in advance to form Fe 2+ – Ascorbic acid complex, effectively inhibits Fe 2+ Oxidized to Fe 3+ , and improve its solubility. Then the complex solution is slowly introduced into the diluted polypeptide solution, and a preliminary reaction is carried out under acidic conditions (pH < 5), which helps Fe 2+ Pre-binding with carboxyl and amino groups in the polypeptide to avoid the formation of ferrous hydroxide precipitation. 2+ A temporary coordination is formed, but as the pH of the reaction system is gradually adjusted to neutral, its coordination ability weakens, and the functional groups with enhanced ionization in the polypeptide are more sensitive to Fe 2+ The affinity of ascorbic acid is increased, thereby promoting the transfer of iron ions from ascorbic acid to peptides, forming a more stable iron-peptide chelate. This "pre-reduction protection-acidic pre-binding-neutral stable complexation" strategy significantly improves the Fe 2+ The high loading rate and complex stability provide a theoretical and technological basis for improving its bioavailability.

[0026] The present invention mainly has the following effects:

[0027] (1) Improve the iron loading rate and improve the stability and dispersibility of the chelate: The present invention introduces a step-by-step chelation reaction process, that is, first mixing ferrous chloride and ascorbic acid to form a stable reduced iron source, and then slowly adding it to the polypeptide solution, and continuing the reaction by adjusting the pH at the end, which significantly improves the binding efficiency between iron ions and polypeptides. The test results show that the prepared polypeptide iron chelate has a more uniform particle size distribution, a higher Zeta potential, and good dispersibility and colloidal stability. Compared with the hemoglobin ferrous chelate peptide disclosed in Chinese patent application No. 202410150997.2 (its maximum iron loading is 58.29g / kg, i.e. 58.29mg / g), the polypeptide iron chelate of the present invention has a maximum iron loading of 154.56mg / g, an increase of more than 160%, which clearly reflects the technical progress of the present invention in iron chelation efficiency.

[0028] (2) It exhibits better solubility and low toxicity in the gastrointestinal environment: Under in vitro simulated gastrointestinal digestion conditions, the prepared chelate showed a significantly higher iron solubility rate than traditional iron supplements (such as ferrous chloride) and maintained good stability in the pH range of 6 to 8. At the same time, Caco-2 cell activity experiments showed that it had no obvious toxicity to cells over a wide concentration range, showing good biocompatibility and lower gastrointestinal irritation, making it suitable for the development of oral iron supplement preparations.

[0029] (3) Green process, no cross-linking agent added, and high biosafety: No cross-linking agent or chemical modifier is added during the preparation process of the present invention, which effectively avoids potential chemical residue problems and further improves the safety of chelates in nutritional or medical products. It is particularly suitable for special populations such as pregnant women and children who need iron supplementation.

[0030] (4) The raw materials are widely available, with the dual value of resource utilization and environmental friendliness: The present invention uses yak blood as raw material, a by-product resource that is often directly discarded in the livestock industry. Through bio-enzymatic hydrolysis and chelation technology, it can achieve high-value utilization, with good sustainability and economic efficiency, which is in line with the development concept of green manufacturing and circular economy. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 A comparison of the effects of step-by-step chelation treatment on peptide-iron chelates;

[0032] Figure 2 This is a comparison chart of particle size distribution and potential measurement results;

[0033] Figure 3 This is the result of the in vitro simulated gastrointestinal digestion test;

[0034] Figure 4 The graph shows the iron solubility test results of polypeptide chelated iron and ferrous chloride under different pH conditions;

[0035] Figure 5 This is the result of CCK-8 cytotoxicity test;

[0036] Figure 6 This is the test result diagram for the optimization of iron loading rate. DETAILED DESCRIPTION

[0037] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below with reference to specific embodiments and drawings, but the embodiments are not intended to limit the present invention.

[0038] The present invention provides a yak whole blood polypeptide iron chelate, as well as its preparation method and application, that addresses the shortcomings of existing iron supplements in terms of solubility, bioavailability, and intestinal irritation. The method first adds yak whole blood to an anticoagulant, repeatedly freeze-thaws to release proteins, and then freeze-dries to obtain a lyophilized powder. The blood is then enzymatically hydrolyzed using the alkaline protease Alcalase to obtain yak whole blood polypeptides. Next, ferrous chloride is mixed with ascorbic acid to improve solubility, and finally, a stepwise chelation reaction is performed to achieve efficient binding of the iron to the polypeptide. The present invention prepares the polypeptide iron chelate through an innovative chelation process. Compared with existing polypeptide iron chelation processes, the chelate produced by this method has a higher iron loading rate and more uniform particle distribution. Compared with commonly used iron supplements, it has greater pH stability, maintains higher solubility in the gastrointestinal environment, and is less irritating to the gastrointestinal tract, making it suitable for the preparation of oral iron supplements. The preparation method of the polypeptide iron chelate provided by the present invention comprises yak blood lyophilized powder, alkaline protease Alcalase, and ferrous chloride. The raw material ingredients are single and contain no chelating agents, ensuring its biosafety.

[0039] In order to achieve the above object, the present invention provides a method for preparing a yak whole blood polypeptide iron chelate, comprising the following steps:

[0040] After adding anticoagulant to yak blood, the blood was repeatedly frozen and thawed to release protein, and then freeze-dried to obtain yak whole blood freeze-dried powder;

[0041] The lyophilized powder is enzymatically hydrolyzed in a water solvent to obtain a yak whole blood polypeptide solution;

[0042] Evenly dissolving ferrous chloride and ascorbic acid in a water solvent to obtain a ferrous chloride solution;

[0043] The yak whole blood polypeptide solution is ultrasonically treated and diluted, and then a ferrous chloride solution is added and stirred for a period of time for a chelation reaction. The pH of the solution is then adjusted to neutral, and the stirring is continued for a chelation reaction. The reactant is then purified to obtain a yak whole blood polypeptide iron chelate.

[0044] The present invention adopts the chelation process, firstly Fe 2+ Mixed with ascorbic acid, it forms a stable reduction complex, which significantly improves the solubility of ferrous iron. Secondly, the initial chelation reaction is carried out under acidic conditions, which effectively avoids the 2+ Finally, the pH of the reaction system is adjusted to neutral to complete the stable formation of the chelate. The present invention greatly improves the iron loading rate and the stability of the chelate by controlling the chelation conditions in stages.

[0045] Wherein, the volume ratio of the yak blood to the anticoagulant is 5 to 10:1;

[0046] Each freeze-thaw cycle involves freezing the yak blood at -30°C to -20°C for 12 to 36 hours, then thawing it in a 36°C to 38°C water bath until it is completely dissolved. The freeze-thaw cycle is repeated 3 to 5 times.

[0047] The freeze-drying temperature is -85 to -75°C and the time is 8 to 24 hours.

[0048] The enzymatic hydrolysis process includes: evenly dispersing the lyophilized powder in an aqueous solvent, and then adding alkaline protease Alcalase for enzymatic hydrolysis; wherein the enzymatic hydrolysis time is 1 to 5 hours, the enzymatic hydrolysis temperature is 30 to 70° C., the enzyme amount is 1 to 5% of the volume, and the mass concentration of the lyophilized powder evenly dispersed in the aqueous solvent is 10 to 50 mg / mL.

[0049] After the enzymatic hydrolysis is completed, the method further includes: heating the enzymatic hydrolysis product in a 100° C. water bath for 10 to 20 minutes, rapidly cooling the product in an ice bath, centrifuging the solution, taking the supernatant, and filtering the solution using a 0.22 μm filter to obtain a yak whole blood polypeptide solution.

[0050] The mass ratio of the ferrous chloride to ascorbic acid is 1-3:1. After mixing, the mixture is sealed and placed at room temperature for 3-5 hours.

[0051] After ultrasonic treatment, the polypeptide solution is added with a ferrous chloride solution, and the chelation reaction is carried out by stirring, wherein the concentration of the polypeptide in the chelation reaction is 1 to 5 mg / mL; the mass ratio of ferrous chloride to polypeptide is 1:2 to 1:10; the reaction is stirred at 20 to 60° C., and the chelation reaction time is 60 to 120 minutes;

[0052] The ultrasonic treatment includes an ultrasonic amplitude of 20% to 40% and an ultrasonic time of 5 to 20 minutes.

[0053] After the initial chelation reaction, the pH of the solution is adjusted to neutral, and the chelation reaction is continued by stirring, including: adjusting the pH to neutral using 0.5-1M NaOH, and continuing the stirring reaction at 20-60°C for 30-60 minutes.

[0054] Purification process, including:

[0055] The solution after the chelation reaction is stirred continuously and collected in a dialysis bag with a molecular weight cutoff of 100Da to 500Da. Deionized water is used as the dialysis medium and dialyzed at 2 to 6°C for 24 to 48 hours. The dialysate is replaced every 4 to 6 hours until the unbound iron ions and impurities are completely removed. After the dialysis is completed, the dialysate is frozen and transferred to a vacuum freeze dryer for freeze drying.

[0056] The present invention adopts the method of dialysis+freeze drying in the purification process to effectively remove unchelated iron ions and low-molecular impurities, ensuring the acquisition of high-purity iron chelates.

[0057] Exemplarily, a method for preparing a yak whole blood polypeptide iron chelate comprises the following steps:

[0058] S1. Yak blood processing: Blood is collected from healthy, artificially raised yaks. After adding an anticoagulant and mixing, the blood is repeatedly frozen and thawed to release protein. Cell debris is removed by centrifugation, and the supernatant is collected and freeze-dried to obtain yak whole blood freeze-dried powder.

[0059] The detailed process of S1 yak blood processing is as follows: healthy yaks are used as raw materials, yak blood is obtained by slaughtering, and the yak blood is mixed with an anticoagulant (20-60 mg / mL sodium citrate) at a volume ratio of 5:1 to 10:1, and transported at low temperature through a cold chain. The yak blood is divided into small vials and frozen at -20°C for 24 hours. The yak blood is taken out and bathed in a 37°C water bath until completely dissolved, and then frozen at -20°C. This is repeated three times. The supernatant is collected by centrifugation at 8000g for 20 minutes at 4°C, and then frozen at -80°C for ≥8 hours. After it is completely frozen, it is placed in a vacuum freeze dryer and freeze-dried to obtain yak whole blood freeze-dried powder.

[0060] S2. Enzymatic hydrolysis: Dissolve the yak whole blood freeze-dried powder in water and add alkaline protease Alcalase for enzymatic hydrolysis. After the reaction is completed, heat to inactivate the enzyme, centrifuge to remove the residue, and filter to sterilize to obtain a yak whole blood polypeptide solution.

[0061] The detailed process of the S2 enzymatic hydrolysis is as follows: the mass concentration of the yak blood freeze-dried powder is 10-50 mg / mL, the amount of enzyme added is 1%-5% by volume, the enzymatic hydrolysis time is 1h-5h, and the enzymatic hydrolysis temperature is 30°C-70°C. Stirring is maintained under these conditions. After the enzymatic hydrolysis is completed, the solution is placed in a 100°C water bath for heating for 10-20 minutes, quickly placed in an ice bath for rapid cooling, the solution is centrifuged, the supernatant is taken, and filtered using a 0.22μm filter to obtain a yak whole blood polypeptide solution.

[0062] S3. Peptide treatment: The obtained peptide solution is subjected to ultrasonic treatment to improve the structural distribution.

[0063] The optimized ultrasonic treatment of the S3 polypeptide includes an ultrasonic amplitude of 20% to 40% and an ultrasonic time of 5 to 20 minutes.

[0064] S4. Preparation of iron source solution: Mix and dissolve ferrous chloride and ascorbic acid in a certain mass ratio to improve the stability and solubility of ferrous chloride.

[0065] The mass ratio of the S4 ferrous chloride and ascorbic acid solution is 1 to 3:1. After mixing, seal and place at room temperature for 3 to 5 hours.

[0066] S5. First stage chelation: dilute the polypeptide obtained in step S3, slowly add the ferrous chloride solution prepared in step S4 dropwise, and stir the reaction under constant temperature.

[0067] The ferrous chloride used in the first step of preparing the S5 yak whole blood polypeptide and ferrous chloride chelate is obtained by S4 operation, and the polypeptide used is obtained by S3. The concentration of the polypeptide in the chelation reaction is 1-5 mg / mL, and the mass ratio of ferrous chloride to polypeptide is 1:2-1:10. The ferrous chloride solution is slowly added to the polypeptide solution, and the reaction is stirred at 20-60° C. The chelation reaction time is 60-120 min.

[0068] S6. Second stage chelation: Adjust the pH of the solution obtained in step S5 to neutral and continue stirring the reaction.

[0069] The second step of preparing the S6 yak whole blood polypeptide and ferrous chloride chelate is to adjust the pH of the solution after the reaction in step S5 to 7 using 0.5-1M NaOH, and continue to stir the reaction at 20-60°C for 30-60 minutes.

[0070] S7. Purification: The chelation reaction product is dialyzed to remove unbound iron and impurities, and then freeze-dried to obtain the yak whole blood polypeptide iron chelate.

[0071] In the step S7, the solution after the reaction is collected and placed in a dialysis bag with a molecular weight cutoff of 100Da to 500Da, and deionized water is used as the dialysis medium. The dialysis is performed at 2 to 6°C for 24 to 48 hours, and the dialysis fluid is replaced every 4 to 6 hours until the unbound iron ions and impurities are completely removed. After the dialysis is completed, the dialysis fluid is frozen and transferred to a vacuum freeze dryer for freeze drying.

[0072] A second aspect of the present invention provides a yak whole blood polypeptide iron chelate.

[0073] A third aspect of the present invention provides a use of a yak whole blood polypeptide iron chelate in the preparation of an iron-supplementing medicine or an iron-supplementing health product.

[0074] It should be noted that the experimental methods used in the present invention are all conventional methods unless otherwise specified; the reagents and materials used are all commercially available unless otherwise specified.

[0075] Example 1

[0076] Yak blood was obtained by slaughtering yaks and mixed with 40 mg / mL sodium citrate in a 9:1 volume ratio for cold chain transportation. The yak blood was aliquoted into small vials and frozen at -20°C for 24 hours. The blood was then removed from the vials and placed in a 37°C water bath until completely dissolved, then frozen again at -20°C. This process was repeated three times. The supernatant was collected by centrifugation at 8000 g for 20 minutes at 4°C and then placed in a -80°C freezer for 8 hours. Once completely frozen, the supernatant was freeze-dried in a vacuum freeze dryer to obtain yak whole blood lyophilized powder for later use.

[0077] The yak whole blood freeze-dried powder was dissolved in water, and an enzyme amount of 5% by volume was added. After the enzymatic hydrolysis was completed, the solution was heated in a 100°C water bath for 10-20 minutes, and then quickly cooled in an ice bath. The solution was centrifuged at 8,000-10,000 rpm for 30-50 minutes, and the supernatant was collected and sterilized by filtration using a 0.22 μm filter to obtain the yak whole blood polypeptide.

[0078] The supernatant was subjected to ultrasonic treatment: ultrasonic amplitude 25%, ultrasonic time 10 min.

[0079] The sonicated peptide solution was diluted 10-fold with water, and ascorbic acid powder was added at a mass ratio of ascorbic acid to peptide of 1:8. Ferrous chloride powder was then added at a mass ratio of 1:4 to the peptide. The pH of the reaction system was adjusted to 7.0 with 0.5 M NaOH solution, and the reaction was carried out under magnetic stirring at a constant temperature of 40°C for 180 minutes. The obtained solution was collected and placed in a dialysis bag with a molecular weight cutoff of 100 Da. The solution was dialyzed at 4°C for 48 hours, and the dialysate was replaced every 6 hours until the impurities were completely removed. After the dialysis was completed, the liquid was collected to obtain a peptide chelated sample.

[0080] Peptide content was determined using the biuret method: 1 mL of each solution was mixed with 1 mL of 10% trichloroacetic acid and centrifuged at 10,000 g for 10 min. 1 mL of the resulting supernatant was mixed with 2 mL of biuret reagent and the absorbance of the mixture was measured at 540 nm. A standard curve was constructed using Gly-Gly-Tyr-Arg as described above, and the peptide content was calculated based on the standard curve.

[0081] It should be noted that the method adopted in Example 1 does not allow ascorbic acid and ferrous chloride to be mixed and dissolved first, nor does it perform chelation in steps during the chelation reaction.

[0082] Example 2

[0083] Yak blood was obtained by slaughtering yaks and mixed with 40 mg / mL sodium citrate in a 9:1 volume ratio for cold chain transportation. The yak blood was aliquoted into small vials and frozen at -20°C for 24 hours. The blood was then removed from the vials and placed in a 37°C water bath until completely dissolved, then frozen again at -20°C. This process was repeated three times. The supernatant was collected by centrifugation at 8000 g for 20 minutes at 4°C and then placed in a -80°C freezer for 8 hours. Once completely frozen, the supernatant was freeze-dried in a vacuum freeze dryer to obtain yak whole blood lyophilized powder for later use.

[0084] The yak whole blood freeze-dried powder was dissolved in water, and an enzyme amount of 5% by volume was added. After the enzymatic hydrolysis was completed, the solution was heated in a 100°C water bath for 10-20 minutes, and then quickly cooled in an ice bath. The solution was centrifuged at 8,000-10,000 rpm for 30-50 minutes, and the supernatant was collected and sterilized by filtration using a 0.22 μm filter to obtain the yak whole blood polypeptide.

[0085] The supernatant was subjected to ultrasonic treatment: ultrasonic amplitude 25%, ultrasonic time 10 min.

[0086] Ferrous chloride and ascorbic acid were mixed and dissolved in a mass ratio of 2:1, sealed and placed at room temperature for 4 h.

[0087] The sonicated peptide solution was diluted 10-fold with water. A mixed solution of ferrous chloride and ascorbic acid was added to the diluted peptide solution at a mass ratio of 1:4 between ferrous chloride and peptide. The pH was adjusted to 7 using 0.5M NaOH, and the reaction was stirred at 40°C for 180 minutes. The resulting solution was collected and placed in a dialysis bag with a molecular weight cutoff of 100 Da. Dialysis was performed at 4°C for 48 hours, with the dialysate replaced every 6 hours until the impurities were completely removed. After dialysis was complete, the liquid was collected to obtain a peptide chelate sample.

[0088] It should be noted that in Example 2, ascorbic acid and ferrous chloride are mixed and dissolved, and then no stepwise chelation is performed during the chelation reaction. The comparison between Example 1 and Example 2 illustrates the importance of mixing and dissolving ascorbic acid and ferrous chloride first in the present invention.

[0089] Example 3

[0090] Yak blood was obtained by slaughtering yaks and mixed with 40 mg / mL sodium citrate in a 9:1 volume ratio for cold chain transportation. The yak blood was aliquoted into small vials and frozen at -20°C for 24 hours. The blood was then removed from the vials and placed in a 37°C water bath until completely dissolved, then frozen again at -20°C. This process was repeated three times. The supernatant was collected by centrifugation at 8000 g for 20 minutes at 4°C and then placed in a -80°C freezer for 8 hours. Once completely frozen, the supernatant was freeze-dried in a vacuum freeze dryer to obtain yak whole blood lyophilized powder for later use.

[0091] The yak blood freeze-dried powder was dissolved in water, and an enzyme amount of 5% by volume was added. After the enzymatic hydrolysis was completed, the solution was heated in a 100°C water bath for 10-20 minutes, and then quickly cooled in an ice bath. The solution was centrifuged at 8,000-10,000 rpm for 30-50 minutes, and the supernatant was collected and sterilized by filtration using a 0.22 μm filter to obtain yak whole blood polypeptides.

[0092] The supernatant was subjected to ultrasonic treatment: ultrasonic amplitude 25%, ultrasonic time 10 min.

[0093] Ferrous chloride and ascorbic acid were mixed and dissolved in a mass ratio of 2:1, sealed and placed at room temperature for 4 h.

[0094] The sonicated peptide solution was diluted 10-fold with triple-distilled water. A mixed solution of ferrous chloride and ascorbic acid was added to the diluted peptide solution at a 1:4 weight ratio of ferrous chloride to peptide. The reaction was stirred at 40°C for 120 minutes. Following the reaction, the pH was adjusted to 7 using 0.5-1M NaOH with slow dropwise addition and stirring. The reaction was continued at 40°C for 60 minutes. The resulting solution was collected and placed in a dialysis bag with a molecular weight cutoff of 100 Da. Dialysis was performed at 4°C for 48 hours, with the dialysate replaced every 6 hours until the impurities were completely removed. After dialysis, the liquid was collected to obtain the peptide-chelated iron sample.

[0095] Example 4

[0096] The same as implementation case 3, except that the mass ratio of ferrous chloride to polypeptide is 1:2.

[0097] Example 5

[0098] The same as implementation case 3, except that the mass ratio of ferrous chloride to polypeptide is 1:6.

[0099] Example 6

[0100] The same as implementation case 3, except that the mass ratio of ferrous chloride to polypeptide is 1:8.

[0101] Example 7

[0102] The same as implementation case 3, except that the mass ratio of ferrous chloride to polypeptide is 1:10.

[0103] Comparative Example 1

[0104] Use ferrous chloride, a commonly used iron supplement on the market.

[0105] In order to illustrate the advantages of the present invention, the following relevant performance tests are provided, which are described in conjunction with the accompanying drawings. The polypeptide chelated iron prepared in the examples is subjected to relevant performance tests as follows.

[0106] Molecular weight distribution, dispersion performance, potential index, and iron content testing: This experiment aims to determine the molecular weight distribution range and uniformity of the polypeptide chelated iron obtained using an innovative chelation process. This test was performed on the polypeptide chelated iron complexes of Examples 1, 2, and 3 of the present invention. The testing method is as follows: dilute the samples of Examples 1, 2, and 3 with triple-distilled water, use a UV spectrophotometer to measure the absorption wavelength from 200 nm to 800 nm, and use a sample concentration with an absorbance of 0.5 to 1 to test the molecular weight distribution and dispersion performance. The instruments used are a laser particle size analyzer and a zeta potential analyzer; and use a ferrozine colorimetric kit to determine the iron content.

[0107] Experimental results: Figure 1 、 2 As shown in Table 1, A corresponds to Example 1, B corresponds to Example 2, and C corresponds to Example 3. Figure 1 It is obvious that the solutions of A and B without step-by-step chelation treatment are turbid, while the solutions after step-by-step chelation treatment are clear, showing smaller and more uniform particle size, and their potential shows strong stability. In addition, their iron content is also significantly improved. All indicators of C are greater than those of B and C, indicating that the step-by-step chelation treatment process effectively improves the surface properties and loading capacity of the sample.

[0108] Table 1 Analysis of particle size, potential and iron content

[0109]

[0110] In vitro simulated gastrointestinal digestion test: This experiment aims to test the iron solubility rate of polypeptide iron chelates in a gastrointestinal digestion environment. This test was performed on the polypeptide iron chelate of Example 3 of the present invention and Comparative Example 1, wherein after the dialysis of the polypeptide iron chelate of Example 3 was completed, the dialysate was frozen and transferred to a vacuum freeze dryer for freeze drying to obtain a polypeptide iron chelate freeze-dried powder. The test method is as follows: prepare simulated gastric fluid: 1 mL of 0.1 M hydrochloric acid is mixed with 40 mg of pepsin, and the pH is adjusted to 2.0; prepare simulated intestinal fluid: 12 mg of bile salts and 2 mg of trypsin are mixed and dissolved in 1 mL of 0.1 M NaHCO3, and the pH is adjusted to 7.5; weigh 10 mg of the freeze-dried powder of the polypeptide iron chelate of Example 3 and the powder of Comparative Example 1 and dissolve them in 10 mL of triple-distilled water. The polypeptide iron chelate of Example 3 and the solution of Comparative Example 1 were placed in a 40°C water bath and shaken for 30 minutes. The pH was then adjusted to 2.0 using 0.1M HCl. After adding simulated gastric fluid at an enzyme-to-substrate mass ratio of 1:100, the mixture was placed in a 37°C water bath and samples were taken at 0, 30, 60, and 90 minutes of digestion. After simulated gastric fluid digestion, the pH was adjusted to 6.8 using NaHCO3 and simulated intestinal fluid was added. After an enzyme-to-substrate mass ratio of 1:100, samples were taken at 0, 10, 30, 60, 90, and 150 minutes of digestion. All samples were immediately placed in a 100°C water bath and heated for 15 minutes to stop enzyme activity. The supernatant was then centrifuged at 8,000g for 10 minutes, and the iron content was determined using a ferrozine colorimetric kit.

[0111] Fe (%) = supernatant Fe (mg) / total Fe (mg) × 100%

[0112] Experimental results: Figure 3 As shown, C corresponds to the polypeptide iron chelate lyophilized powder of Example 3, and D corresponds to Example 1. The in vitro simulated gastrointestinal digestion test shows that the polypeptide iron chelate shows significant solubility during intestinal digestion, while ferrous chloride has poor solubility in the intestine, indicating that the polypeptide iron chelate can effectively improve the bioavailability of iron.

[0113] Solubility test of Fe under different pH conditions: This experiment aims to test the solubility of Fe of the sample under pH conditions to verify stability. This test was performed on the polypeptide iron chelate of Example 3 of the present invention and Comparative Example 1, wherein after the dialysis of the polypeptide iron chelate of Example 3 was completed, the dialysate was frozen and transferred to a vacuum freeze dryer for freeze drying to obtain a polypeptide iron chelate lyophilized powder. The test method is as follows: 10 mg of the polypeptide iron chelate of Example 3 and 10 mg of the powder of Comparative Example 1 were dissolved in 10 mL of pH 2, 3, 4, 5, 6, 7, and 8 buffer respectively, shaken in a 37°C water bath for 10 minutes, centrifuged at 5,000g for 10 minutes, collected the supernatant, and the iron content was determined using a ferrozine colorimetric kit.

[0114] Fe (%) = supernatant Fe (mg) / total Fe (mg) × 100%

[0115] Experimental results: Figure 4 As shown, C corresponds to the polypeptide iron chelate lyophilized powder of Example 2, and D corresponds to Example 1. As the pH gradually increases, the solubility rates of the two gradually decrease. The solubility rate of the polypeptide iron chelate iron is significantly higher than that of ferrous chloride in a pH range of 6 to 8, indicating that the polypeptide iron chelate has stronger stability in a higher pH environment and can better maintain its dissolved state.

[0116] CCK-8 toxicity test: This experiment is designed to test the toxic effect of iron on Caco-2 cells. The test was conducted on the polypeptide iron chelate freeze-dried powder of Example 3 of the present invention and Comparative Example 1. The test method is as follows: Caco-2 cells were plated at 1×10 4 Cells / well were seeded into 96-well plates using a culture medium consisting of MEM, 20% fetal bovine serum, 2mM L-glutamine, and 1% double-antibody. The cells were cultured in a 37°C incubator for 24 hours. The polypeptide iron chelate of Example 3 and the solution of Comparative Example 1 were prepared using the culture medium and sterilized by filtration using a 0.22 μm filter in a clean bench. 2+ The concentration of CCK-8 was 100 μg / mL, which was then serially diluted with fresh culture medium to obtain 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL, 1.5 μg / mL, and 0.78 μg / mL, respectively. The old culture medium was removed from the cultured cells, and the cells were gently washed with PBS. Samples with different iron ion concentrations were added sequentially to a 96-well plate, with 100 μL added to each well. The cells were cultured in a 37°C incubator for 24 hours. CCK-8 reagent treatment solution was prepared: 100 μL of culture medium was added to 10 μL of CCK-8 reagent. After incubation, the culture medium was discarded, the cells were gently washed with PBS, and 110 μL of CCK-8 reagent treatment solution was added to each well. After incubation at 37°C for 2 hours, the absorbance of the solution was measured at 450 nm.

[0117] Cell activity = (A1-A0) / (A2-A0)×100%

[0118] A0 is the absorbance of the well containing culture medium and CCK-8 reagent, A1 is the absorbance of the well containing cells, culture medium, CCK-8 and test substance, and A2 is the absorbance of the well containing cells, culture medium and CCK-8.

[0119] The experimental results are as follows Figure 5 As shown, the left figure corresponds to Example 3, and the right figure corresponds to Example 1. 2+With the increase of concentration, the polypeptide iron chelate showed lower cytotoxicity and good biocompatibility; while the Fe 2+ At higher concentrations, such as 50 μg / mL and above, cytotoxicity increased significantly, leading to a significant decrease in cell survival. These results indicate that the peptide iron chelate maintains low cytotoxicity over a wide concentration range, demonstrating its potential advantages and good safety in iron supplementation therapy and other biomedical applications.

[0120] Iron Loading Rate Optimization Test: This experiment aimed to test the effect of different peptide-to-iron chelation ratios on iron loading and to identify the optimal chelation ratio. This test was performed on Examples 3, 4, 5, 6, and 7 of the present invention. The testing method was as follows: samples were collected after dialysis in each case and the peptide content was determined using the biuret method, and the iron content was determined using a ferrozine colorimetric assay kit.

[0121] Iron loading rate (mg / g) = M0 / M1

[0122] M0 is the mass of ferrous iron in the polypeptide iron chelate (mg); M1 is the mass of the polypeptide iron chelate (g).

[0123] Experimental results: The ratio of ferrous chloride to polypeptide of 1:4 showed the best iron loading rate of 154.56 mg / g. Excessive ferrous chloride could not be completely combined in the polypeptide chelation reaction, and some iron ions may not be effectively chelated due to the excess, forming precipitation, thereby reducing the iron loading efficiency; when the concentration of ferrous chloride is too low, the available iron ions in the chelation reaction are reduced, resulting in insufficient iron loading.

[0124] In summary, the present invention provides an innovative method for preparing a yak whole blood polypeptide and ferrous chloride chelate. The raw materials include yak blood freeze-dried powder, alkaline protease, and ferrous chloride. The raw material composition is simple and chelating agent-free, ensuring its biosafety. Compared with existing polypeptide chelation iron processes, the complex produced by this method has a higher iron loading rate and more uniform particle distribution. Compared with commonly used iron supplements, it has stronger pH stability, maintains higher solubility in the gastrointestinal environment, and is less irritating to the gastrointestinal tract, making it suitable for the preparation of oral iron supplements.

[0125] The present invention describes preferred embodiments and their effects. However, those skilled in the art, once informed of the basic inventive concept, may make additional changes and modifications to these embodiments. Therefore, it is intended that the appended claims be interpreted to include the preferred embodiments as well as all changes and modifications that fall within the scope of the invention.

[0126] Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that several improvements and modifications may be made without departing from the principles and spirit of the invention, and that these improvements and modifications should also be considered as within the scope of protection of the present invention, which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing yak whole blood polypeptide iron chelate, characterized in that: The following steps are involved: After adding anticoagulant to yak blood, the blood was repeatedly frozen and thawed to release protein, and then freeze-dried to obtain yak whole blood freeze-dried powder; The lyophilized powder is enzymatically hydrolyzed in a water solvent to obtain a yak whole blood polypeptide solution; Evenly dissolving ferrous chloride and ascorbic acid in a water solvent to obtain a ferrous chloride solution; The yak whole blood polypeptide solution is ultrasonically treated and diluted, and then a ferrous chloride solution is added and stirred for a chelation reaction. The pH of the solution is then adjusted to neutral, and the stirring is continued for a chelation reaction. The reactant is then purified to obtain a yak whole blood polypeptide iron chelate.

2. The method for preparing the yak whole blood polypeptide iron chelate according to claim 1, characterized in that: The volume ratio of the yak blood to the anticoagulant is 5 to 10:1; Each freeze-thaw cycle involves freezing the yak blood at -30°C to -20°C for 12 to 36 hours, then thawing it in a 36°C to 38°C water bath until it is completely dissolved. The freeze-thaw cycle is repeated 3 to 5 times. The freeze-drying temperature is -85 to -75°C and the time is 8 to 24 hours.

3. The method for preparing the yak whole blood polypeptide iron chelate according to claim 1, characterized in that: The enzymatic hydrolysis process includes: evenly dispersing the lyophilized powder in an aqueous solvent, and then adding alkaline protease Alcalase for enzymatic hydrolysis; wherein the enzymatic hydrolysis time is 1 to 5 hours, the enzymatic hydrolysis temperature is 30 to 70° C., the enzyme amount is 1 to 5% of the volume, and the mass concentration of the lyophilized powder evenly dispersed in the aqueous solvent is 10 to 50 mg / mL.

4. The method for preparing the yak whole blood polypeptide iron chelate according to claim 1, characterized in that: After the enzymatic hydrolysis is completed, the method further includes: heating the enzymatic hydrolysis product in a 100° C. water bath for 10 to 20 minutes, rapidly cooling the product in an ice bath, centrifuging the solution, taking the supernatant, and filtering the solution using a 0.22 μm filter to obtain a yak whole blood polypeptide solution.

5. The method for preparing the yak whole blood polypeptide iron chelate according to claim 1, characterized in that: The mass ratio of the ferrous chloride to ascorbic acid is 1-3:

1. After mixing, the mixture is sealed and placed at room temperature for 3-5 hours.

6. The method for preparing the yak whole blood polypeptide iron chelate according to claim 1, characterized in that: When adding ferrous chloride solution and stirring for chelation reaction, the concentration of the polypeptide in the chelation reaction is 1 to 5 mg / mL; the mass ratio of ferrous chloride to polypeptide is 1:2 to 1:10; stirring the reaction at 20 to 60° C., and the chelation reaction time is 60 to 120 minutes; The ultrasonic treatment includes an ultrasonic amplitude of 20% to 40% and an ultrasonic time of 5 to 20 minutes.

7. The method for preparing yak whole blood polypeptide iron chelate according to claim 1, characterized in that: The pH of the solution is adjusted to neutral, and the stirring is continued to perform a chelating reaction, including: adjusting the pH to neutral using 0.5-1 M NaOH, and continuing to stir the reaction at 20-60° C. for 30-60 minutes.

8. The method for preparing yak whole blood polypeptide iron chelate according to claim 1, characterized in that: Purification process, including: The solution after the chelation reaction is stirred continuously and collected in a dialysis bag with a molecular weight cutoff of 100Da to 500Da. Deionized water is used as the dialysis medium and dialyzed at 2 to 6°C for 24 to 48 hours. The dialysate is replaced every 4 to 6 hours until the unbound iron ions and impurities are completely removed. After the dialysis is completed, the dialysate is frozen and transferred to a vacuum freeze dryer for freeze drying.

9. A yak whole blood polypeptide iron chelate prepared by the method according to any one of claims 1 to 8.

10. Use of the yak whole blood polypeptide iron chelate according to claim 9 in the preparation of iron-supplementing medicines or iron-supplementing health products.

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