Deer blood peptide integrated ferrous blood supplement and preparation method thereof

Abstract

The application discloses a kind of deer blood peptide integrated ferrous blood tonic and preparation method thereof, using alkaline protease and flavourzyme step-by-step enzymolysis deer blood, and 500-1500Da target deer blood peptide is obtained by combining chelation fitting value determination;The target deer blood peptide is integrated chelation with ferrous salt under the condition of avoiding oxygen, controlling pH and constant temperature, and is purified by membrane separation, vacuum drying and finished product review to obtain blood tonic.The method can reduce free iron residue and ferrous oxidation risk, improve gastric acid stability, reconstitution stability and iron absorption utilization rate.

Description

Technical Field

[0001] This invention relates to the field of functional nutritional products and bioactive peptide chelated iron preparations. More specifically, this invention relates to the process of directional enzymatic hydrolysis of deer blood peptides, ferrous ion chelation stabilization, and preparation of iron supplements, and particularly to a deer blood peptide-integrated ferrous iron supplement and its preparation method. Background Technology

[0002] Iron deficiency anemia and iron insufficiency are common nutritional health problems, and supplementing with absorbable iron sources is an important way to improve iron nutritional status. Among existing iron supplements, ferrous salts such as ferrous sulfate, ferrous chloride, and ferrous lactate are widely used due to their wide availability and low cost. However, ordinary ferrous salts, in a free ionic state, easily convert to ferric iron (Fe3+) in the acidic environment of the stomach and under oxidative conditions, or form insoluble precipitates with food components, leading to a decrease in iron absorption and utilization. At the same time, free ferrous ions can irritate the gastrointestinal mucosa, easily causing discomfort such as nausea, abdominal pain, and diarrhea, affecting long-term adherence to use.

[0003] To improve iron absorption and reduce gastrointestinal irritation, existing technologies have developed products such as amino acid chelated iron and peptide chelated iron. These products reduce the proportion of free iron through coordination or chelation, thus improving the stability of iron in the digestive tract. However, existing peptide chelated iron typically uses hydrolyzed peptides from common animal and plant proteins. These peptides have a wide molecular weight distribution and unstable coordination sites, leading to inconsistent chelation rates and gastric acid stability. Some products still experience free iron release, ferrous oxidation, or precipitation in the acidic environment of the stomach, making it difficult to simultaneously achieve stability, absorbability, and low irritation.

[0004] Deer blood is rich in protein and nitrogen-containing active ingredients, which can be hydrolyzed to obtain small-molecule deer blood peptides. These small-molecule peptides possess good solubility and coordination potential, making them suitable as chelating carriers for ferrous ions. However, existing deer blood peptide preparation processes mostly rely on single-enzymatic hydrolysis or fixed-time enzymatic hydrolysis, lacking synergistic control over the relationship between the degree of hydrolysis, the proportion of target molecular weight, the degree of free amino exposure, and subsequent chelation capacity. This can easily lead to problems such as peptides being too large, too small, or unevenly distributed. If the molecular weight of the deer blood peptide is too large, it may affect the uniformity of absorption and chelation; if the molecular weight is too small, it may be difficult to form a stable cyclic chelate structure, affecting the ferrous ion protection effect.

[0005] Therefore, existing technologies still lack a method for preparing deer blood peptide-integrated ferrous iron supplements that can combine precise enzymatic hydrolysis of deer blood peptides, ferrous chelation, free iron control, gastric acid stability verification, and parameter feedback correction. How to obtain a deer blood peptide-integrated ferrous iron supplement with suitable molecular weight, stable coordination ability, low oxidation and low free iron residue under gastric acid conditions, and suitable for industrial production, is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] To overcome the shortcomings of existing ferrous iron supplements, such as easy oxidation and precipitation in the acidic environment of the stomach, strong gastrointestinal irritation, and low iron absorption and utilization, this invention provides a deer blood peptide-integrated ferrous iron supplement and its preparation method. This invention uses deer blood peptides within a specific molecular weight range as coordination carriers for ferrous ions, and utilizes enzymatic hydrolysis fit values, chelation rates, free iron content, and stomach acid stability verification results to form a closed-loop process. This establishes a continuous feedback relationship between deer blood peptide preparation, ferrous chelation, purification and drying, and finished product determination, thereby obtaining a deer blood peptide-integrated ferrous iron supplement with good stomach acid stability, low free iron content, and suitability for intestinal release and absorption.

[0007] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing a deer blood peptide-integrated ferrous hematinic agent includes deer blood pretreatment, stepwise enzymatic hydrolysis, deer blood peptide fractionation, ferrous chelation, separation and purification, vacuum drying, and finished product verification. First, fresh deer blood is filtered, centrifuged, and separated into protein solutions to obtain a deer blood protein solution to be enzymatically hydrolyzed. Then, stepwise enzymatic hydrolysis is performed using alkaline protease and flavor protease, and the degree of hydrolysis, molecular weight distribution, proportion of small peptides, and free amino content are collected online to calculate the chelation suitability value of the deer blood peptide. When the chelation suitability value meets a preset range, the enzymatic hydrolysis is terminated, and the deer blood peptide hydrolysate is obtained. Further, through enzyme inactivation, filtration, concentration, drying, and fractionation, a target deer blood peptide powder with a molecular weight of 500-1500 Da is obtained. Then, the target deer blood peptide powder is prepared into a deer blood peptide solution, and ferrous salt is added at a mass ratio of deer blood peptide to ferrous salt (3-5:1). An oxygen-avoided chelation reaction is carried out under conditions of pH 5.0-7.0 and temperature 40-60℃ to obtain crude deer blood peptide-integrated ferrous salt. Finally, after membrane separation purification and vacuum drying, the initial product of deer blood peptide integrated ferrous iron supplement was obtained. The product was then verified by gastric acid stability, free iron content and reconstitution stability to determine whether it could be exported as a finished product.

[0008] By adopting the above technical solution, this invention does not simply mix deer blood peptides with ferrous salts, but controls the enzymatic hydrolysis endpoint based on the molecular weight of deer blood peptides, the degree of peptide exposure, and the chelation coordination value, so that the peptides entering the chelation reaction have a high ferrous coordination ability; at the same time, the endpoint of the chelation process is determined by the free iron content and chelation rate, and the gastric acid stability verification results are used to correct the enzymatic hydrolysis endpoint, the amount of ferrous salt added, and the chelation pH value, thereby forming a quality closed loop in the preparation process.

[0009] In a preferred embodiment, the deer blood pretreatment step includes: taking fresh deer blood, filtering to remove blood clots and mechanical impurities, centrifuging at 3000-5000 r / min for 10-20 min to separate deer blood protein solution. After centrifugation, the protein concentration and soluble protein recovery rate of the deer blood protein solution are detected. When the soluble protein recovery rate is lower than a preset value, the centrifugation time or centrifugation speed is readjusted to ensure the stability of the subsequent enzymatic hydrolysis substrate concentration.

[0010] By adopting the above technical solutions, the impact of serum impurities, coagulation residues, and insoluble impurities on enzymatic hydrolysis efficiency can be reduced, resulting in a more stable protein concentration and reaction uniformity of the enzymatic hydrolysis substrate.

[0011] In a preferred embodiment, the stepwise enzymatic hydrolysis step employs a combination of alkaline protease and flavor protease, preferably in a mass ratio of 2:1, with the total enzyme addition amounting to 2%-4% of the deer blood protein mass. The first stage involves enzymatic hydrolysis at 48-52℃ for 1.5-2.5 hours to open the main structure of the deer blood protein. The second stage involves synergistic enzymatic hydrolysis at 33-37℃ for 0.8-1.2 hours to further release small molecule peptides and improve the exposure of peptide coordination sites. During the enzymatic hydrolysis process, the degree of hydrolysis, molecular weight distribution, and free amino content are monitored, and the appropriate chelate ratio for the deer blood peptides is calculated.

[0012] By adopting the above technical solution, the problems of excessively wide molecular weight distribution or insufficient proportion of active peptides caused by single enzymatic hydrolysis can be avoided. This allows deer blood peptides to have both the characteristics of small molecules suitable for absorption and active sites that can form coordination chelate structures with ferrous ions.

[0013] In a preferred embodiment, the appropriate chelate ratio of deer blood peptides is determined by the degree of hydrolysis, the proportion of peptides with a molecular weight of 500-1500 Da, the proportion of peptides with a molecular weight less than 1000 Da, and the content of free amino groups. If the hydrolysis is too low, the protein is not fully cleaved, resulting in peptides with larger molecular weights; if the hydrolysis is too high, the peptides are excessively fragmented, which may weaken the ability to form a stable cyclic chelate structure. Therefore, this invention preferably controls the degree of hydrolysis at 25%-28% and ensures that the proportion of peptides with a molecular weight of 500-1500 Da is not less than 80%.

[0014] By adopting the above technical solution, the enzymatic hydrolysis endpoint can be transformed from simple time control to multi-index adaptive control, so that the deer blood peptide preparation result is directly related to the subsequent ferrous chelation ability.

[0015] In a preferred embodiment, the deer blood peptide fractionation step is performed using ultrafiltration membrane or gel filtration. For peptides with a molecular weight greater than 1500 Da, they can be refluxed to the enzymatic hydrolysis stage for further enzymatic hydrolysis; for components with a molecular weight less than 500 Da, they can be discharged as ineffective small molecule impurities; the 500-1500 Da component is retained as the target deer blood peptide powder.

[0016] By adopting the above technical solution, the molecular weight range of deer blood peptides entering the chelation reaction can be more concentrated, which is conducive to improving the chelation rate, reducing free iron residue, and improving the batch stability of the finished product.

[0017] In a preferred embodiment, the ferrous salt includes one or more of ferrous sulfate, ferrous chloride, ferrous lactate, or ferrous gluconate, preferably ferrous sulfate or ferrous lactate. After the deer blood peptide powder is prepared into a 10%-15% (w / w) deer blood peptide solution, ferrous salt is added at a deer blood peptide to ferrous salt mass ratio of (3-5):1, and the amount of ferrous salt added in the next batch is adjusted based on the free iron content and chelation rate of the previous batch.

[0018] By adopting the above technical solution, we can avoid the problem of low iron content due to insufficient ferrous salt addition, and also avoid the problem of increased risk of free ferrous residue and gastrointestinal irritation due to excessive ferrous salt addition.

[0019] In a preferred embodiment, the chelation reaction is carried out for 2-4 hours at a pH of 5.0-7.0 and a temperature of 40-60°C. During the reaction, the free ferrous content, total iron content, pH value of the reaction solution, dissolved oxygen content, and chelation rate are collected. The reaction is considered to have reached its endpoint when the chelation rate reaches 85% or higher and the rate of increase in the chelation rate is lower than a preset growth threshold within a continuous sampling period. If the dissolved oxygen content increases, the risk of ferrous oxidation is reduced by extending the nitrogen purging time or reducing the stirring speed.

[0020] By adopting the above technical solution, the chelation rate can be improved while reducing ferrous oxidation and free iron residue, so that deer blood peptides and ferrous ions can form an integrated structure more stably.

[0021] In a preferred embodiment, the purification and drying step includes membrane separation and vacuum drying. Membrane separation is used to remove unchelated free ferrous ions, ineffective small molecule impurities, and large molecule residues; the vacuum drying temperature is controlled at 50-60°C, and the product is dried until the moisture content is no higher than 5%.

[0022] By adopting the above technical solutions, the risk of free iron ions irritating the gastrointestinal mucosa can be reduced, and the stability of the finished product during storage and reconstitution can be guaranteed.

[0023] In a preferred embodiment, the final product verification includes gastric acid stability verification, free iron content verification, and reconstitution stability verification. For gastric acid stability verification, the sample is placed in simulated gastric acid solution with a pH of 1.2-2.0 and incubated at 36-38°C for 1.5-2.5 hours, and the ferrous oxidation rate and precipitation amount are measured. If the ferrous oxidation rate, free iron content, or precipitation amount does not meet the final product judgment criteria, the deviation information is fed back to the enzymatic hydrolysis, ferrous addition, or chelation reaction steps for parameter correction.

[0024] By adopting the above technical solution, the present invention can form a closed-loop control from deer blood peptide preparation to finished product stability evaluation, which is suitable for batch quality stability control in industrial continuous production.

[0025] The technical effects and advantages of this invention are as follows: 1. This invention establishes a closed-loop control process for the preparation of deer blood peptides, ferrous chelation, and finished product verification. This allows blood tonics to no longer rely on simple mixing or fixed process parameters. Instead, key conditions are dynamically adjusted based on the degree of hydrolysis, molecular weight distribution, chelation rate, and gastric acid stability, thereby reducing the risks of ferrous oxidation, free iron residue, and gastrointestinal irritation.

[0026] 2. This invention uses 500-1500 Da target deer blood peptide as ferrous coordination carrier, and calculates the chelation coordination value based on the degree of hydrolysis, the proportion of target peptide, and the content of free amino groups. This can improve the structural matching between peptide and ferrous ions, making it easier for the finished product to form a stable chelate structure and improving the problem of easy oxidation and precipitation of ordinary ferrous salts in the gastric acid environment.

[0027] 3. This invention overcomes the problems of wide peptide distribution and low proportion of effective small molecule peptides caused by single enzymatic hydrolysis by stepwise enzymatic hydrolysis and molecular weight fractionation control. This makes the deer blood peptide components entering the chelation reaction more concentrated, which is conducive to improving the chelation rate, reducing batch differences, and providing a stable raw material basis for industrial continuous production.

[0028] 4. This invention monitors the free ferrous iron content, chelation rate, pH value and dissolved oxygen content in real time during the chelation reaction, and reduces ferrous iron oxidation by judging the oxygen avoidance reaction and the reaction endpoint, so that more ferrous ions exist in the integrated state of deer blood peptide, thereby reducing the direct stimulation of the digestive tract by free ferrous iron from the source.

[0029] 5. This invention removes unchelated ferrous ions, ineffective small molecule impurities, and large molecule residues through membrane separation purification, and combines it with vacuum drying to control the moisture content, so that the finished product has good reconstitution stability and storage stability, and reduces the risks of precipitation, increase of free iron, and dissociation of chelated structures.

[0030] 6. This invention uses gastric acid stability, free iron content, and reconstituted precipitation as finished product judgment indicators, and feeds back unqualified deviation information to the enzymatic hydrolysis endpoint, ferrous salt addition amount, or chelation pH value for correction, so that the production process can be continuously optimized and a complete quality control chain from deer blood raw material to blood supplement finished product is formed. Attached Figure Description

[0031] Figure 1 This is a flowchart of a method for preparing a deer blood peptide-integrated ferrous iron supplement.

[0032] Figure 2 This is a flowchart of the stepwise enzymatic hydrolysis and deer blood peptide fitting value determination process in this invention.

[0033] Figure 3 This is a flowchart of the ferrous chelation, endpoint control, and purification process in this invention.

[0034] Figure 4 This is a flowchart of the finished product verification and closed-loop parameter correction process in this invention.

[0035] Figure 5 This is a flowchart of the preparation process of the compound formulation in this invention. Detailed Implementation

[0036] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention; equivalent substitutions made to enzymes, ferrous salt sources, membrane separation specifications, drying methods, detection methods, and dosage form excipients without departing from the technical concept of the present invention shall all fall within the scope of protection of the present invention.

[0037] This invention provides a deer blood peptide-integrated ferrous iron supplement and its preparation method. The method uses fresh deer blood as the protein raw material and involves deer blood pretreatment, stepwise enzymatic hydrolysis with compound enzymes, determination of the appropriate chelation ratio of deer blood peptides, grading of target molecular weight peptides, fractional addition of ferrous salts, oxygen-avoided chelation reaction, determination of chelation endpoint, membrane separation and purification, vacuum drying, and final product verification to obtain the deer blood peptide-integrated ferrous iron supplement. Unlike methods that simply perform enzymatic hydrolysis at a fixed time or directly mix peptides with ferrous salts, this invention incorporates the degree of hydrolysis, the proportion of target peptides, the content of free amino groups, the chelation rate, the content of free iron, the ferrous oxidation rate, and the reconstitution stability into a continuous control process, forming a closed-loop process that can be corrected by feedback between the deer blood peptide preparation results and the ferrous chelation effect.

[0038] Reference Figure 1 The overall preparation process of this invention includes, in sequence, deer blood pretreatment, deer blood protein separation, stepwise enzymatic hydrolysis, determination of appropriate chelation ratio for deer blood peptides, enzyme inactivation filtration, concentration and drying, 500-1500 Da deer blood peptide fractionation, deer blood peptide solution preparation, fractional addition of ferrous salt, oxygen-avoided chelation reaction, determination of chelation endpoint, membrane separation and purification, vacuum drying, and finished product verification. When the finished product verification result meets the finished product judgment conditions, the deer blood peptide integrated ferrous iron supplement is output as a finished product. When the finished product verification result does not meet the requirements, parameter feedback correction is performed according to the deviation type, and the correction result is returned to key steps such as stepwise enzymatic hydrolysis, fractional addition of ferrous salt, or oxygen-avoided chelation reaction to improve the consistency of subsequent batches of products.

[0039] Reference Figure 2The stepwise enzymatic hydrolysis and deer blood peptide chelation suitability determination process of the present invention includes: adding alkaline protease and flavor protease to deer blood protein solution, performing enzymatic hydrolysis in the first stage at 48-52℃ to open the main structure of deer blood protein; then performing synergistic enzymatic hydrolysis in the second stage at 33-37℃ to release small molecule active peptides; sampling at regular intervals during enzymatic hydrolysis, and detecting the degree of hydrolysis, the proportion of peptides with a molecular weight of 500-1500 Da, the proportion of peptides with a molecular weight of less than 1000 Da, and the content of free amino acids; then standardizing the above indicators and calculating the deer blood peptide chelation suitability value by weighting; when the suitability value does not reach the preset range, returning to the second stage of synergistic enzymatic hydrolysis; when the suitability value reaches the preset range, immediately performing enzyme inactivation, filtration, concentration, and drying to obtain the target deer blood peptide suitable for ferrous chelation.

[0040] Reference Figure 3 The ferrous chelation, endpoint control, and purification process of this invention includes: preparing a deer blood peptide powder into a deer blood peptide solution, adding ferrous salt in stages, and creating a low-oxygen environment by nitrogen replacement; adjusting the pH of the system to 5.0-7.0, and then performing a constant-temperature stirring chelation reaction at 40-60℃; simultaneously monitoring the free ferrous content, total iron content, dissolved oxygen, and chelation rate during the reaction; determining that the chelation endpoint has been reached when the chelation rate reaches 85% or more and the increase in chelation rate within a continuous sampling period is lower than a preset threshold, thus obtaining crude deer blood peptide with integrated ferrous iron; subsequently purifying using an ultrafiltration membrane to remove unchelated free ferrous ions, ineffective small molecule impurities, and large molecule residues; and ending the purification process and vacuum drying when the free iron content is lower than a preset limit to obtain the initial blood-replenishing agent.

[0041] Reference Figure 4 The finished product verification and closed-loop parameter correction process of this invention includes: incubating the initial hematinic agent sample under simulated gastric acid conditions and detecting the ferrous oxidation rate, precipitation amount, free iron content, and reconstitution state; when the above indicators meet the finished product judgment conditions, the finished product is output; when the finished product judgment conditions are not met, the deviation type is analyzed; if the deviation is manifested as high free iron, the amount of ferrous salt added and the membrane separation purification time are corrected; if the deviation is manifested as low chelation rate, the enzymatic hydrolysis endpoint and the proportion of target peptides are corrected; if the deviation is manifested as high ferrous oxidation rate or precipitation amount, the oxygen avoidance conditions, pH value, and stirring intensity are corrected. The corrected parameters are written into the process database and used for the next batch of process parameters.

[0042] Reference Figure 5After obtaining the deer blood peptide-integrated ferrous iron supplement, edible excipients or nutritional ingredients can be added according to the application form of the product, and it can be made into liquid beverage, solid beverage, granules or oral liquid. During the compounding process, the pH value, water content and heat treatment conditions of the system should be controlled to avoid the destruction of the integrated structure formed by deer blood peptide and ferrous ions. For liquid dosage forms, the focus is on precipitation observation and free iron verification. For solid dosage forms, the focus is on testing the moisture content and reconstitution stability.

[0043] Example 1: Closed-loop preparation of deer blood peptide-integrated ferrous hematoxylin and eosinogen This embodiment provides a method for preparing a deer blood peptide-integrated ferrous hematoxylin and eosinogen.

[0044] First, 1 kg of fresh deer blood was taken, and after coarse filtration to remove blood clots and mechanical impurities, it was centrifuged at 4000 r / min for 15 min to obtain deer blood protein solution. The protein concentration and soluble protein recovery rate of the deer blood protein solution were measured; when the protein recovery rate was lower than 90%, the centrifugation time was extended or the centrifugation speed was adjusted before re-separation. In this example, the protein recovery rate was 92%, which met the requirements for subsequent enzymatic hydrolysis.

[0045] Subsequently, the deer blood protein solution was adjusted to a suitable reaction state for enzymatic hydrolysis, and alkaline protease and flavor protease were added at a mass ratio of 2:1, with the total enzyme addition amounting to 3% of the deer blood protein mass. The first stage involved enzymatic hydrolysis at 50℃ for 2 hours to fully develop the main structure of the deer blood protein. The second stage involved cooling to 35℃ and continuing synergistic enzymatic hydrolysis for 1 hour to release a higher proportion of small molecule active peptides. Samples were collected at regular intervals during the enzymatic hydrolysis process to detect the degree of hydrolysis, molecular weight distribution, proportion of small molecule peptides, and free amino acid content.

[0046] In this embodiment, the enzymatic hydrolysis process does not use a fixed hydrolysis time as the sole endpoint, but rather calculates the optimal chelate ratio for deer blood peptides. Specifically, the degree of hydrolysis, the proportion of peptides with a molecular weight of 500-1500 Da, the proportion of peptides with a molecular weight of less than 1000 Da, and the free amino content are standardized and weighted according to preset weights to obtain the optimal chelate ratio for deer blood peptides. When the degree of hydrolysis reaches 25%, the proportion of peptides with a molecular weight of 500-1500 Da is not less than 80%, the proportion of peptides with a molecular weight of less than 1000 Da reaches approximately 85%, and the free amino content is within a preset range, the optimal chelate ratio for deer blood peptides is deemed to have met the requirements. At this point, the temperature is immediately raised to 90°C for 10 minutes to inactivate the enzyme and terminate the enzymatic hydrolysis reaction.

[0047] After enzyme inactivation, the enzymatic hydrolysate was filtered to remove enzymatic residue, then concentrated under vacuum and spray-dried to obtain deer blood peptide powder. Subsequently, the deer blood peptide powder reconstituted solution was fractionated using an ultrafiltration membrane, retaining the 500-1500 Da fraction as the target deer blood peptide powder; fractions larger than 1500 Da were refluxed to the enzymatic hydrolysis section for further enzymatic hydrolysis, while fractions smaller than 500 Da were discharged as ineffective small molecule impurities. In this example, the yield of the target deer blood peptide was 78%.

[0048] Next, the target deer blood peptide powder was dissolved in purified water to prepare a 12% (w / w) deer blood peptide solution. Ferrous sulfate was added at a deer blood peptide to ferrous sulfate mass ratio of 4:1, with stirring maintained during the addition process. To reduce the risk of ferrous oxidation, nitrogen gas was introduced into the reaction vessel before the chelation reaction began to purge and maintain the reaction system in a low-oxygen state. Subsequently, the pH was adjusted to 6.0 using a citrate-disodium hydrogen phosphate buffer system, and the reaction was carried out at a constant temperature of 50°C with stirring for 3 hours.

[0049] During the reaction, the free ferrous content, total iron content, pH value of the reaction solution, dissolved oxygen content, and chelation rate are collected in real time. When the chelation rate reaches 85% or higher and the increase in chelation rate within a continuous sampling period is lower than a preset growth threshold, the chelation reaction is determined to have reached a stable endpoint. In this embodiment, the chelation rate is 88%. If the free ferrous content is detected to be too high, the amount of ferrous salt added or the chelation reaction time is extended in the next batch. If the ferrous oxidation rate is detected to be too high, the inert gas replacement is strengthened or the stirring shear intensity is reduced in the next batch.

[0050] After chelation, crude deer blood peptide-integrated ferrous iron was obtained. The crude product was then purified by ultrafiltration to remove unchelated free ferrous ions, ineffective small molecule impurities, and large molecule residues. During purification, the conductivity of the permeate, the free iron content, and the color change were monitored. Purification was terminated when the free iron content was below a preset limit. Subsequently, the product was vacuum dried at 55°C until the moisture content was 4.2%, yielding the initial product of deer blood peptide-integrated ferrous iron supplement.

[0051] Finally, the initial product was verified as a finished product. The sample was placed in simulated gastric acid at pH 1.5 and incubated at 37°C for 2 hours. The ferrous oxidation rate, precipitation amount, and resolution state were measured. In this example, the ferrous oxidation rate was 3.2%, with no obvious precipitation or flocculation, and the free iron content was below the preset limit, thus it was determined to be a qualified finished product. The verification result was also written into the process database and used as reference data for the next batch of ferrous salt addition, enzymatic hydrolysis endpoint, and chelation pH correction.

[0052] Example 2: Adjustment of Ferrous Salt Addition Ratio This embodiment is basically the same as Embodiment 1, except that: the mass ratio of deer blood peptide to ferrous sulfate is 3:1, the chelation reaction temperature is 45℃, and the reaction time is 4 hours; due to the relatively high amount of ferrous salt added, the focus during the reaction is on monitoring the free ferrous residue and the growth trend of the chelation rate. The reaction is terminated when the chelation rate reaches 86% and the free iron content is not higher than the preset limit.

[0053] Testing showed that the chelation rate of the hematologic product obtained in this embodiment was 86%, and the yield of the target deer blood peptide was 76%. This embodiment illustrates that when the amount of ferrous salt added is increased, the stability of the finished product can be maintained by extending the reaction time and strengthening membrane separation and purification to remove free iron. This is suitable for product solutions with high target iron content but still requiring control of gastrointestinal irritation risks.

[0054] Example 3: High Chelation Efficiency Example This embodiment is basically the same as Embodiment 1, except that: the mass ratio of deer blood peptide to ferrous sulfate is 5:1, the chelation reaction temperature is 60℃, and the reaction time is 2h. Since the relative content of deer blood peptide is relatively high, the coordination sites are more sufficient, which is conducive to reducing the residue of free iron.

[0055] Testing showed that the hematologic replenishment product obtained in this embodiment had a chelation rate of 90%, a target deer blood peptide yield of 79%, and a finished product moisture content of 4.5%. This embodiment demonstrates that when the proportion of deer blood peptide is increased, the chelation efficiency can be improved by appropriately increasing the reaction temperature and shortening the reaction time, while reducing the risk of free iron caused by excessive ferrous salts.

[0056] Example 4: Closed-loop parameter correction example In industrial continuous production, if a batch of finished products shows an iron oxidation rate exceeding 5% in simulated gastric acid testing or slight precipitation after resolution, the system will feed this deviation information back to the preparation process.

[0057] Specifically, if the deviation is mainly manifested as an increase in free iron content, the amount of ferrous salt added and the membrane separation and purification time will be corrected first; if the deviation is mainly manifested as a low chelation rate, the enzymatic hydrolysis endpoint will be corrected first to increase the proportion of the 500-1500 Da target peptide; if the deviation is mainly manifested as an increase in ferrous oxidation rate, the oxygen avoidance conditions, pH value and stirring intensity will be corrected first. The corrected process parameters will be written into the process database and used as a reference for the enzymatic hydrolysis endpoint, ferrous salt addition amount, chelation pH value and oxygen avoidance conditions in the next batch of production.

[0058] Through this closed-loop correction, the preparation method can adjust key process parameters in reverse according to the finished product indicators, rather than relying on a fixed formula for long-term production, thereby improving the consistency of finished products under different batches of deer blood raw materials.

[0059] Example 5: Compound Formulation Example After obtaining the deer blood peptide-integrated ferrous iron supplement, it can be further formulated into liquid beverages, solid beverages, granules, or oral liquids with edible excipients or nutritional ingredients. Optional excipients include one or more of the following: erythritol, concentrated hawthorn juice, concentrated wolfberry juice, double-petal red rose extract, longan powder, astragalus powder, angelica powder, tangerine peel powder, yam powder, or konjac powder. The above excipients are mainly used to improve taste, dispersibility, and compliance, without changing the technical essence of deer blood peptide-integrated ferrous iron as the core iron-supplementing active ingredient.

[0060] During the compounding process, the pH value, water content, and heat treatment conditions of the system should be controlled to avoid damage to the chelate structure. If a liquid dosage form is used, precipitation observation and verification of free iron content should be performed. If a solid dosage form is used, the moisture content and reconstitution stability should be controlled. Once the relevant indicators meet the requirements, the finished compound preparation can be obtained.

[0061] Comparative Example 1: Conventional Ferrous Iron Supplements Commercially available ferrous sulfate supplements were used as comparative example 1, without deer blood peptide integration or peptide chelation treatment; this comparative example is used to illustrate the shortcomings of iron supplements in the free ferrous ion state in terms of gastric acid environment stability, gastrointestinal irritation, and iron absorption and utilization rate.

[0062] Comparative Example 2: Ordinary Deer Blood Peptide Chelated Iron Supplement A chelated iron supplement was prepared using common deer blood peptides with a molecular weight greater than 1500 Da and by enzymatic hydrolysis with a single alkaline protease at 50°C for 3 hours. In this comparative example, the peptide yield was 42%. This comparative example illustrates that without target molecular weight control, stepwise enzymatic hydrolysis, and chelation matching value determination, the yield of deer blood peptides, the proportion of small molecule peptides, and the chelation stability will all be affected.

[0063] Performance testing Samples from Example 1, Comparative Example 1, and Comparative Example 2 were selected for performance comparison testing. Three parallel samples were set up in each group, and the average value of the test results was taken.

[0064] In the test of the deer blood peptide preparation process effect, the peptide yield of Example 1 was 78%, and the peptide yield of Comparative Example 2 was 42%; the proportion of small molecule peptides in Example 1, i.e., components with a molecular weight of less than 1000 Da, was 85%, and that of Comparative Example 2 was 45%; the degree of hydrolysis in Example 1 was 25%, and that of Comparative Example 2 was 12%. It can be seen that the present invention can significantly improve the yield of target small molecule deer blood peptides and the proportion of peptides suitable for ferrous chelation by stepwise enzymatic hydrolysis with alkaline protease and flavor protease, combined with the determination of the appropriate chelation ratio of deer blood peptides.

[0065] In the gastric acid stability test, each group of samples was placed in simulated gastric acid solution at pH 1.5 and incubated at 37°C for 2 hours. The ferrous ion oxidation rate was then measured. The results showed that the ferrous oxidation rate of Example 1 was 3.2%, that of Comparative Example 1 was 41.5%, and that of Comparative Example 2 was 18.7%. These results indicate that the deer blood peptide-integrated ferrous blood supplement obtained in this invention has a good ferrous ion protection effect under simulated gastric acid environment and can reduce the risk of ferrous ion oxidation and precipitation.

[0066] In the gastrointestinal irritation test, 60 SPF-grade Kunming mice were randomly divided into 3 groups of 20 each. Each group was administered samples of Example 1, Comparative Example 1, and Comparative Example 2 with equal iron content via gavage. Gastrointestinal reactions were observed after 7 consecutive days of gavage. In Example 1, no diarrhea, vomiting, or loss of appetite was observed; the mice were in good spirits, and there was no congestion or edema of the gastrointestinal mucosa, with an adverse reaction rate of 0%. In Comparative Example 1, 14 mice experienced diarrhea and loss of appetite, and significant congestion and edema of the gastrointestinal mucosa, with an adverse reaction rate of 70%. In Comparative Example 2, 8 mice experienced mild gastrointestinal discomfort, with an adverse reaction rate of 40%. These results indicate that by forming an integrated structure with ferrous ions and reducing the free iron content, deer blood peptides can help reduce the direct irritation of the gastrointestinal mucosa by free ferrous ions.

[0067] In the iron absorption and utilization rate test, a mouse model of iron deficiency anemia was used. After 14 days of oral administration, the hemoglobin content and serum iron content of the mice were measured, and the iron absorption rate was calculated. The results showed that the iron absorption rate of Example 1 was 63%, and the hemoglobin content recovered to 98% of the normal level; the iron absorption rate of Comparative Example 1 was 12%, and the hemoglobin content recovered to 35% of the normal level; the iron absorption rate of Comparative Example 2 was 28%, and the hemoglobin content recovered to 52% of the normal level. These results indicate that the product of this invention has a higher iron absorption and utilization level compared with ordinary ferrous salts and ordinary deer blood peptide chelated iron products.

[0068] In the product stability test, each group of samples was placed in an environment of 40℃ and 75% humidity for 3 months to test the chelation stability of the products. The results showed that the chelation structure of Example 1 remained intact, the free iron ion content was no more than 1%, and the stability was 99%; Comparative Example 1 was completely oxidized and failed, and the free iron ion content was 98%; Comparative Example 2 had a partially dissociated chelation structure, the free iron ion content was 22%, and the stability was 78%. These results indicate that the present invention, through target peptide fractionation, oxygen-avoiding chelation, membrane separation purification, and finished product verification, is beneficial to improving the chelation structure stability of hematologic supplement products under storage conditions.

[0069] In summary, Examples 1 to 5, as well as Comparative Examples 1 and 2, collectively demonstrate that the present invention, through stepwise enzymatic hydrolysis, chelation and matching value determination, target deer blood peptide grading, oxygen-avoiding chelation, endpoint control, membrane separation and purification, and finished product verification, forms a complete preparation system from deer blood raw material to hematologic supplement finished product and then to compound formulation. This system can improve the yield of target small molecule deer blood peptide and ferrous chelation rate, reduce free iron residue and ferrous oxidation risk, improve gastric acid stability, gastrointestinal tolerance, iron absorption and utilization rate, and product storage stability, making it suitable for industrial mass production.

[0070] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a deer blood peptide-integrated ferrous hematinic agent, characterized in that, Includes the following steps: S1. Fresh deer blood is filtered, centrifuged, and separated into protein solutions. The protein concentration, initial pH value, and soluble protein recovery rate of the deer blood protein solution are collected. The amount of compound enzyme to be added is determined based on the protein concentration to obtain the deer blood protein solution to be enzymatically hydrolyzed. S2. Add alkaline protease and flavor protease to the deer blood protein solution to be enzymatically hydrolyzed for stepwise enzymatic hydrolysis. During the enzymatic hydrolysis process, collect the degree of hydrolysis, molecular weight distribution, proportion of small molecule peptides and free amino content. Calculate the appropriate chelation value of deer blood peptide according to the degree of hydrolysis, the proportion of target molecular weight and the degree of free amino exposure. Terminate the enzymatic hydrolysis when the appropriate chelation value of deer blood peptide reaches the preset appropriate range to obtain deer blood peptide enzymatic hydrolysate. S3. The deer blood peptide hydrolysate is subjected to enzyme inactivation, filtration, concentration and drying, and deer blood peptide components with a molecular weight of 500-1500 Da are sieved to obtain the target deer blood peptide powder. S4. Dissolve the target deer blood peptide powder in purified water to form a deer blood peptide solution. Determine the amount of ferrous salt to be added based on the appropriate chelation ratio of deer blood peptide, the concentration of deer blood peptide solution and the target iron content. Add the ferrous salt in stages to the deer blood peptide solution to obtain the chelation reaction solution. S5. The pH of the chelation reaction solution is adjusted, the temperature is kept constant and the chelation reaction is carried out in an oxygen-free manner. The free ferrous content, total iron content, pH value of the reaction solution and chelation rate are collected in real time. The chelation reaction is judged to have reached the endpoint based on the decrease of free ferrous content and the increase of chelation rate, and the crude product of deer blood peptide integrated ferrous iron is obtained. S6. The crude deer blood peptide-integrated ferrous iron product is purified by membrane separation to remove unchelated free ferrous ions, ineffective small molecule impurities and large molecule residues, and the retained components are vacuum dried to obtain the initial product of deer blood peptide-integrated ferrous iron supplement. S7. Verify the gastric acid stability, free iron content, and reconstitution stability of the initial product of deer blood peptide integrated ferrous iron supplement. When the ferrous iron oxidation rate, free iron content, and reconstitution precipitation all meet the finished product judgment conditions, output the finished product of deer blood peptide integrated ferrous iron supplement. When any indicator does not meet the finished product judgment conditions, feed back the corresponding deviation information to the enzymatic hydrolysis endpoint of S2, the ferrous salt addition amount of S4, or the chelation pH value of S5 for parameter correction in the next batch.

2. The method for preparing a deer blood peptide-integrated ferrous hematinic agent according to claim 1, characterized in that, The appropriate chelation value of deer blood peptide in S2 is determined as follows: the degree of hydrolysis, the proportion of peptides with a molecular weight of 500-1500 Da, the proportion of peptides with a molecular weight of less than 1000 Da, and the content of free amino groups are converted into standardized indicators and weighted according to preset weights. When the degree of hydrolysis is 25%-28%, the proportion of peptides with a molecular weight of 500-1500 Da is not less than 80%, the proportion of peptides with a molecular weight of less than 1000 Da is not less than 75%, and the content of free amino groups is within the preset amino exposure range, the appropriate chelation value of deer blood peptide is determined to have reached the preset fit range.

3. The method for preparing a deer blood peptide-integrated ferrous hematinic agent according to claim 1, characterized in that, The mass ratio of alkaline protease to flavor protease in S2 is 2:1, and the total enzyme addition is 2%-4% of the deer blood protein. The first stage of enzymatic hydrolysis is carried out at 48-52℃ for 1.5-2.5h, and then the temperature is lowered to 33-37℃ for the second stage of synergistic enzymatic hydrolysis for 0.8-1.2h. The second stage of enzymatic hydrolysis time is adjusted according to the hydrolysis degree increase rate and the target molecular weight component increase rate.

4. The method for preparing a deer blood peptide-integrated ferrous hematinic agent according to claim 1, characterized in that, When sieving and retaining deer blood peptide components with a molecular weight of 500-1500 Da in S3, at least one of ultrafiltration membrane or gel filtration is used for classification; when the molecular weight of a component greater than 1500 Da exceeds the preset upper limit, the component is refluxed to the enzymatic hydrolysis section for further enzymatic hydrolysis; when the molecular weight of a component less than 500 Da exceeds the preset upper limit, the component is discharged as an invalid small molecule impurity.

5. A method for preparing a deer blood peptide-integrated ferrous hematinic agent according to claim 1, characterized in that, The ferrous salt in S4 is derived from one or more of ferrous sulfate, ferrous chloride, ferrous lactate, or ferrous gluconate; the mass ratio of the target deer blood peptide powder to the ferrous salt is (3-5):1, the mass concentration of the deer blood peptide solution is 10%-15%, and the amount of ferrous salt added is adjusted according to the free iron content and chelation rate in the previous batch of finished products.

6. A method for preparing a deer blood peptide-integrated ferrous hematinic agent according to claim 1, characterized in that, In S5, the pH value of the chelation reaction is controlled at 5.0-7.0, the reaction temperature is 40-60℃, and the reaction time is 2-4h. When the free ferrous content decreases continuously and the chelation rate reaches more than 85%, the chelation reaction is determined to have entered the stable stage. When the increase in chelation rate is lower than the preset growth threshold within the continuous sampling period, the chelation reaction is terminated.

7. A method for preparing a deer blood peptide-integrated ferrous hematinic agent according to claim 1, characterized in that, When performing the oxygen-avoiding chelation reaction in S5, nitrogen or inert gas is introduced into the reaction vessel to create a low-oxygen reaction environment, and the dissolved oxygen content is controlled within a preset range. When the dissolved oxygen content exceeds the preset range, the stirring speed is reduced or the inert gas replacement time is extended to reduce the risk of ferrous oxidation.

8. A method for preparing a deer blood peptide-integrated ferrous hematinic agent according to claim 1, characterized in that, In S6, membrane separation and purification is performed using an ultrafiltration membrane with a molecular weight cutoff of 500-1500 Da. The degree of removal of unchelated ferrous iron and small molecule impurities is judged by conductivity, free iron content, and color change of the permeate. When the free iron content is not higher than the preset free iron limit, purification ends and vacuum drying begins.

9. A method for preparing a deer blood peptide-integrated ferrous hematinic agent according to claim 1, characterized in that, The gastric acid stability verification in S7 involves placing the deer blood peptide-integrated ferrous iron supplement in simulated gastric acid solution with a pH of 1.2-2.0 and incubating it at 36-38℃ for 1.5-2.5 hours, then detecting the ferrous oxidation rate and precipitation amount. When the ferrous oxidation rate is not higher than 5%, the precipitation amount is not higher than the preset precipitation limit, and there is no obvious flocculation after reconstitution, it is determined to meet the gastric acid stability requirements.

10. A deer blood peptide-integrated ferrous hematinic agent, characterized in that, The deer blood peptide integrated ferrous iron supplement prepared by any one of claims 1-9 comprises a cyclic coordination chelate structure formed by deer blood peptide with a molecular weight of 500-1500 Da and ferrous ions, with a chelation rate of not less than 85%, a moisture content of not more than 5%, a free iron content of not more than a preset free iron limit, and is able to maintain a low ferrous oxidation rate under simulated gastric acid environment.

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