Donkey-hide gelatin peptide-iron chelate preparation and preparation method thereof

Abstract

The invention belongs to the technical field of functional food, and particularly relates to a donkey-hide gelatin peptide-iron chelate preparation and a preparation method thereof. The preparation method comprises the following steps: adding colla corii asini powder into water, and melting by heat to obtain a colla corii asini stock solution; adding protease into the colla corii asini stock solution for enzymolysis to obtain colla corii asini enzymatic hydrolysate; bOC-L-cysteine-curcumin, tetrabutylammonium bromide and iodine are added into the colla corii asini enzymatic hydrolysate, a sodium hydroxide solution with the concentration of 0.1 mol / L is adopted for adjusting the pH value, then a reaction is conducted, and a colla corii asini enzymatic hydrolysate retreatment solution is prepared; ascorbic acid is added into the donkey-hide gelatin enzymolysis retreatment liquid, the pH is adjusted with a 10% hydrochloric acid solution, then ferrous chloride is added, centrifugation is carried out after chelation, nanofiltration treatment is carried out, and a nanofiltration concentrated solution is a donkey-hide gelatin peptide-iron chelate; and uniformly stirring and mixing the colla corii asini peptide-iron chelate and genkwanin to obtain the colla corii asini peptide-iron chelate

Description

Technical Field

[0001] This invention belongs to the field of functional food technology, and particularly relates to a donkey-hide gelatin peptide-iron chelate preparation and its preparation method. Background Technology

[0002] Iron deficiency anemia (IDA), the most prevalent nutritional deficiency disease worldwide, has undergone four generations of evolution in its iron supplementation treatment strategies: from early ferrous sulfate and ferrous lactate, to ferrous glycine, and now to heme iron, polysaccharide iron, and peptide iron preparations. Despite the increasing efficacy of iron supplementation, traditional iron supplements still face three major challenges: low iron absorption and utilization, strong gastrointestinal irritation, and the risk of pro-inflammatory reactions caused by iron overload. Iron, as an essential trace element, is also a double-edged sword; it can catalyze free radical reactions, exacerbating intestinal oxidative stress and inflammatory responses. Therefore, how to achieve adequate iron supplementation while effectively inhibiting iron overload and related inflammatory responses has become a key technological bottleneck in the development of fourth-generation iron supplements.

[0003] Colla Corii Asini, also known as donkey-hide gelatin, has long been hailed as a "holy medicine for nourishing blood" in traditional medicine. Modern research shows that the small-molecule active peptides obtained through enzymatic hydrolysis can interact with Fe... 2+ This forms a structurally stable donkey-hide gelatin peptide-iron chelate (EPI). This type of complex combines the advantages of "ferrous chelate peptides," including high absorption efficiency, low gastrointestinal irritation, and low free iron release, thus providing an ideal carrier for developing a new generation of functional iron supplements that combine highly effective blood-tonifying function with excellent intestinal safety.

[0004] Curcumin, due to the conjugated backbone of polyphenolic hydroxyl groups and β-diketones in its molecular structure, can form stable five- or six-membered ring chelates with iron ions in the gastrointestinal environment, thereby significantly inhibiting free radical chain reactions and lipid peroxidation. However, its hydrophobic aromatic ring structure easily drives intermolecular π-π stacking, resulting in poor aqueous dispersion and weak mucosal permeability, which severely restricts its bioavailability and limits its practical application potential in iron chelation and antioxidation. Therefore, it is essential to modify it to broaden its applications. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, one of the objectives of this invention is to provide a simple method for preparing a donkey-hide gelatin peptide-iron chelate preparation.

[0006] One of the objectives of this invention is achieved through the following technical solution: A method for preparing a donkey-hide gelatin peptide-iron chelate preparation includes the following steps: S1. Preparation of donkey-hide gelatin solution The gelatin powder is dissolved in water, cooled, and the volume is adjusted to obtain gelatin stock solution. S2. Preparation of donkey-hide gelatin enzymatic hydrolysate Add protease to the gelatin stock solution for enzymatic hydrolysis, filter after hydrolysis to obtain gelatin hydrolysate; S3. Preparation of enzymatic hydrolysis and reprocessing solution for donkey-hide gelatin BOC-L-cysteine-curcumin, tetrabutylammonium bromide, and iodine were added to the enzymatic hydrolysate of donkey-hide gelatin. The pH was adjusted with 0.1 mol / L sodium hydroxide solution, and then the reaction was carried out. After the reaction was completed, the mixture was ultrafiltered to obtain the enzymatic hydrolysate reprocessing solution of donkey-hide gelatin. The structural formula of BOC-L-cysteine-curcumin is as follows:

[0007] S4. Preparation of donkey-hide gelatin peptide-iron chelate Add ascorbic acid to the enzymatic hydrolysis and reprocessing solution of donkey-hide gelatin, adjust the pH with 10% hydrochloric acid solution, then add ferrous chloride, chelate and centrifuge, and then perform nanofiltration. The nanofiltration concentrate is the donkey-hide gelatin peptide-iron chelate. S5. Preparation of donkey-hide gelatin peptide-iron chelate formulation The donkey-hide gelatin peptide-iron chelate is stirred and mixed evenly with genkwa extract, then sieved, sterilized, and packaged.

[0008] Furthermore, in step S1, the mass ratio of donkey-hide gelatin powder to water is 1:(20-30); the melting temperature is 60-90℃, and the time is 15-30 minutes.

[0009] Furthermore, in step S2, the amount of protease added is 3-5% of the donkey-hide gelatin powder in the donkey-hide gelatin stock solution; the protease is trypsin or bromelain; the enzymatic hydrolysis conditions are: temperature 40-50℃, rotation speed 45-50 r / min, pH 7-7.5; and the enzymatic hydrolysis time is 3-5 h.

[0010] Furthermore, in step S3, the amounts of BOC-L-cysteine-curcumin, tetrabutylammonium bromide, and iodine added are 8-15%, 5-12%, and 3-5% of the gelatin enzymatic hydrolysate, respectively; the pH is 9-10; and the reaction time is 3-5 hours.

[0011] Furthermore, the preparation method of the BOC-L-cysteine-curcumin is as follows: BOC-L-cysteine, curcumin, and triethylamine are added to a tetrahydrofuran aqueous solution for reaction. After the reaction is completed, the mixture is purified to obtain the BOC-L-cysteine-curcumin.

[0012] Furthermore, the molar ratio of curcumin, BOC-L-cysteine, and triethylamine is 1:(1.2-1.6):(1.2-1.6); the volume ratio of tetrahydrofuran to water in the tetrahydrofuran aqueous solution is 1:5; the reaction temperature is 20-25℃, and the reaction time is 3-5h.

[0013] Furthermore, in step S4, the ratio of the amount of donkey-hide gelatin enzymatic hydrolysis reprocessing solution, ascorbic acid, and ferrous chloride is 1 mL: (0.001-0.002) g: (0.003-0.006) g; the pH is adjusted to 4-5; and the chelation temperature is 20-25℃ and the time is 20-30 min.

[0014] Furthermore, in step S5, the mass ratio of the donkey-hide gelatin peptide-iron chelate to genkwa extract is (10-20):(0.1-0.5); and the stirring temperature is 65-75℃.

[0015] The second objective of this invention is to provide a donkey-hide gelatin peptide-iron chelate preparation, which has a positive effect on the treatment of iron deficiency anemia in mice.

[0016] The second objective of this invention is achieved by the following technical solution: A donkey-hide gelatin peptide-iron chelate preparation was prepared using the above-mentioned preparation method for donkey-hide gelatin peptide-iron chelate preparation.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The donkey-hide gelatin peptide of this invention is rich in active amino acid residues. Its amino group can form an amide bond with the carboxyl group of BOC-L-cysteine-curcumin through dehydration condensation, thereby enhancing the donkey-hide gelatin peptide's ability to chelate ferrous ions. The prepared donkey-hide gelatin peptide-iron chelate retains the original carboxyl and amino groups' complexing ability with ferrous ions. BOC-L-cysteine-curcumin, through its 1,3-diketone (β-diketone) and terminal phenolic hydroxyl oxygen-coordination site, forms an O,O-bidentate six-membered ring chelate with ferrous ions, increasing the chelation rate of donkey-hide gelatin peptides for ferrous ions and inhibiting the free iron-catalyzed Fenton reaction. In vitro DPPH experiments also confirmed the enhanced antioxidant activity of the complex. This process does not require the introduction of an exogenous synthetic carrier, and can simultaneously improve the water solubility of iron and the mucosal permeability of the complex system, thereby enhancing the bioavailability of iron. This provides a green, efficient, and scalable precision nutrition strategy for oral intervention in iron deficiency anemia.

[0018] 2. This invention further combines the donkey-hide gelatin peptide-iron chelate with genkwa extract to form a formulation. The aromatic rings of genkwa extract and curcumin are stacked π-π, and their phenolic hydroxyl groups form hydrogen bonds and van der Waals force networks with the carboxyl and amino groups of the donkey-hide gelatin peptide; spatially shielding the central iron ion reduces the exchange with endogenous ligands (such as phosphate and bicarbonate ions), giving the chelate higher kinetic stability in the physiological environment. Detailed Implementation

[0019] The present invention will be further described below with reference to specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. Specific conditions not specified in the embodiments shall be performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all reagents or instruments used are conventional products obtained through commercial channels.

[0020] Preparation Example 1 This preparation example provides a BOC-L-cysteine-curcumin, prepared by the following method:

[0021] Following a reaction ratio of 1 mmol:1.5 mmol:1.5 mmol:2 mL, BOC-L-cysteine, curcumin, and triethylamine were added to a tetrahydrofuran aqueous solution (v / v, 1:5). The mixture was reacted at 22 °C for 4 h. After the reaction was complete, the solvent was removed by freeze-drying, and the solution was then slurried with dichloromethane to obtain BOC-L-cysteine-curcumin. The NMR and mass spectrometry results of BOC-L-cysteine-curcumin are as follows: 1 HNMR (C 29 H 35 NO 10 S,DMSO- d6 ,400MHz)δ:12.39(s,1H),9.91(s,1H),9.52(s,1H),7.60(d,1H),7.42(s,1H),7.11(d,1H),6.97(d,1H),6.90(d,1H), 6.80-6.71(m,4H),4.75(t,1H),4.12(s,2H),4.05(t,1H),3.85(s,3H),3.77(s,3H),3.25-2.81(m,4H),1.43(s,9H).MS m / z 590.20(M+1),Found:590.20. Preparation Example 2 This preparation example provides a BOC-L-cysteine-curcumin, prepared by the following method: According to the ratio of curcumin, BOC-L-cysteine, triethylamine, and tetrahydrofuran aqueous solution 1 mmol: 1.2 mmol: 1.2 mmol: 2 mL, BOC-L-cysteine, curcumin, and triethylamine were added to tetrahydrofuran aqueous solution (v / v, 1:5), and reacted at 20 °C for 5 h. After the reaction was completed, the solvent was removed by freeze drying, and then the mixture was slurried with dichloromethane to obtain BOC-L-cysteine-curcumin. The NMR and mass spectrometry results of BOC-L-cysteine-curcumin were the same as those in Preparation Example 1.

[0022] Preparation Example 3 This preparation example provides a BOC-L-cysteine-curcumin, prepared by the following method: According to the ratio of curcumin, BOC-L-cysteine, triethylamine, and tetrahydrofuran aqueous solution 1 mmol: 1.6 mmol: 1.6 mmol: 2 mL, BOC-L-cysteine, curcumin, and triethylamine were added to tetrahydrofuran aqueous solution (v / v, 1:5), and reacted at 25 °C for 3 h. After the reaction was completed, the solvent was removed by freeze drying, and then the mixture was slurried with dichloromethane to obtain BOC-L-cysteine-curcumin. The NMR and mass spectrometry results of BOC-L-cysteine-curcumin were the same as those in Preparation Example 1.

[0023] Example 1 This embodiment provides a method for preparing a donkey-hide gelatin peptide-iron chelate preparation, comprising the following steps: S1. Preparation of donkey-hide gelatin solution According to the mass ratio of donkey-hide gelatin powder to water of 1:25, 75g of donkey-hide gelatin powder was added to water and melted at 70℃ for 20 minutes to obtain donkey-hide gelatin melted solution. After cooling the donkey-hide gelatin melted solution to room temperature, water was added to make up to 1500mL and shaken well to obtain donkey-hide gelatin stock solution. S2. Preparation of donkey-hide gelatin enzymatic hydrolysate The amount of protease added was 4% of the amount of donkey-hide gelatin powder in the donkey-hide gelatin stock solution. Trypsin was added to 1000 mL of the donkey-hide gelatin stock solution and enzymatically hydrolyzed for 4 h at a temperature of 45℃, a rotation speed of 47 r / min, and a pH of 7.2. After the enzymatic hydrolysis was completed, the donkey-hide gelatin hydrolysate was treated with an ultrafiltration membrane with a molecular weight cutoff of 3000 Da. The resulting filtrate was then treated with an ultrafiltration membrane with a molecular weight cutoff of 1000 Da to obtain the donkey-hide gelatin hydrolysate. S3. Preparation of enzymatic hydrolysis and reprocessing solution for donkey-hide gelatin The amounts of BOC-L-cysteine-curcumin, tetrabutylammonium bromide, and iodine added were 10%, 7%, and 4% of the amount of the donkey-hide gelatin enzymatic hydrolysate, respectively. BOC-L-cysteine-curcumin, tetrabutylammonium bromide, and iodine (from Preparation Example 1) were added to 100 mL of the donkey-hide gelatin enzymatic hydrolysate. The pH was adjusted to 9.5 using 0.1 mol / L sodium hydroxide solution, and the mixture was reacted at room temperature for 4 hours. After the reaction was completed, the reaction solution was first treated with an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, and the resulting filtrate was then treated with an ultrafiltration membrane with a molecular weight cutoff of 1000 Da to obtain the donkey-hide gelatin enzymatic hydrolysate reprocessing solution. S4. Preparation of donkey-hide gelatin peptide-iron chelate According to the ratio of 1 mL : 0.0015 g : 0.006 g of enzymatic hydrolysis reprocessing solution of donkey-hide gelatin, ascorbic acid and ferrous chloride, add ascorbic acid to 500 mL of enzymatic hydrolysis reprocessing solution of donkey-hide gelatin, adjust the pH to 4.5 with 10% hydrochloric acid solution, and then add ferrous chloride. Chelate at 22℃ for 25 min, centrifuge at 4500 r / min for 5 min, and remove unreacted ferrous chloride by nanofiltration. The nanofiltration concentrate is donkey-hide gelatin peptide-iron chelate. S5. Preparation of donkey-hide gelatin peptide-iron chelate formulation Add genkwa extract to the donkey-hide gelatin peptide-iron chelate obtained in S4 at a mass ratio of 1:0.025, stir and mix evenly at 70℃, and then sieve, sterilize and package.

[0024] This embodiment also provides a donkey-hide gelatin peptide-iron chelate preparation, which is prepared using the above-described preparation method.

[0025] Example 2 This embodiment provides a method for preparing a donkey-hide gelatin peptide-iron chelate preparation, comprising the following steps: S1. Preparation of donkey-hide gelatin solution According to the mass ratio of donkey-hide gelatin powder to water of 1:20, 75g of donkey-hide gelatin powder was added to water and melted at 60℃ for 30 minutes to obtain donkey-hide gelatin melted solution. After cooling the donkey-hide gelatin melted solution to room temperature, water was added to make up to 1500mL and shaken well to obtain donkey-hide gelatin stock solution. S2. Preparation of donkey-hide gelatin enzymatic hydrolysate The amount of protease added was 3% of the amount of donkey-hide gelatin powder in the donkey-hide gelatin stock solution. Bromelain was added to 1000 mL of the donkey-hide gelatin stock solution and enzymatically hydrolyzed for 5 h at a temperature of 40℃, a rotation speed of 45 r / min and a pH of 7. After the enzymatic hydrolysis was completed, the donkey-hide gelatin hydrolysate was treated with an ultrafiltration membrane with a molecular weight cutoff of 3000 Da. The resulting filtrate was then treated with an ultrafiltration membrane with a molecular weight cutoff of 1000 Da to obtain the donkey-hide gelatin hydrolysate. S3. Preparation of enzymatic hydrolysis and reprocessing solution for donkey-hide gelatin The amounts of BOC-L-cysteine-curcumin, tetrabutylammonium bromide, and iodine added were 8%, 5%, and 3% of the amount of the donkey-hide gelatin enzymatic hydrolysate, respectively. BOC-L-cysteine-curcumin, tetrabutylammonium bromide, and iodine (from Preparation Example 2) were added to 100 mL of the donkey-hide gelatin enzymatic hydrolysate. The pH was adjusted to 9 using 0.1 mol / L sodium hydroxide solution, and the mixture was reacted at room temperature for 3 hours. After the reaction was completed, the reaction solution was treated with an ultrafiltration membrane with a molecular weight cutoff of 3000 Da. The resulting filtrate was then treated with an ultrafiltration membrane with a molecular weight cutoff of 1000 Da to obtain the donkey-hide gelatin enzymatic hydrolysate reprocessing solution. S4. Preparation of donkey-hide gelatin peptide-iron chelate According to the ratio of 1 mL : 0.001 g : 0.004 g of enzymatic hydrolysis reprocessing solution of donkey-hide gelatin, ascorbic acid and ferrous chloride are added to 500 mL of enzymatic hydrolysis reprocessing solution of donkey-hide gelatin, the pH is adjusted to 4 with 10% hydrochloric acid solution, and then ferrous chloride is added. The mixture is chelated at 20℃ for 30 min, centrifuged at 4500 r / min for 5 min, and unreacted ferrous chloride is removed by nanofiltration. The nanofiltration concentrate is the donkey-hide gelatin peptide-iron chelate. S5. Preparation of donkey-hide gelatin peptide-iron chelate formulation Add genkwa extract to the donkey-hide gelatin peptide-iron chelate obtained in S4 at a mass ratio of 1:0.005, stir and mix evenly at 65℃, and then sieve, sterilize and package.

[0026] This embodiment also provides a donkey-hide gelatin peptide-iron chelate preparation, which is prepared using the above-described preparation method.

[0027] Example 3 This embodiment provides a method for preparing a donkey-hide gelatin peptide-iron chelate preparation, comprising the following steps: S1. Preparation of donkey-hide gelatin solution According to the mass ratio of donkey-hide gelatin powder to water of 1:30, 75g of donkey-hide gelatin powder was added to water and melted at 90℃ for 15 minutes to obtain donkey-hide gelatin melted solution. After cooling the donkey-hide gelatin melted solution to room temperature, water was added to make up to 1500mL and shaken well to obtain donkey-hide gelatin stock solution. S2. Preparation of donkey-hide gelatin enzymatic hydrolysate The amount of protease added was 5% of the amount of donkey-hide gelatin powder in the donkey-hide gelatin stock solution. Trypsin was added to 1000 mL of the donkey-hide gelatin stock solution and enzymatically hydrolyzed for 3 h at a temperature of 50℃, a rotation speed of 50 r / min, and a pH of 7.5. After the enzymatic hydrolysis was completed, the donkey-hide gelatin hydrolysate was treated with an ultrafiltration membrane with a molecular weight cutoff of 3000 Da. The resulting filtrate was then treated with an ultrafiltration membrane with a molecular weight cutoff of 1000 Da to obtain the donkey-hide gelatin hydrolysate. S3. Preparation of enzymatic hydrolysis and reprocessing solution for donkey-hide gelatin The amounts of BOC-L-cysteine-curcumin, tetrabutylammonium bromide, and iodine added were 15%, 12%, and 5% of the amount of the donkey-hide gelatin enzymatic hydrolysate, respectively. BOC-L-cysteine-curcumin, tetrabutylammonium bromide, and iodine (preparation example 3) were added to 100 mL of the donkey-hide gelatin enzymatic hydrolysate. The pH was adjusted to 10 using 0.1 mol / L sodium hydroxide solution, and the mixture was reacted at room temperature for 5 h. After the reaction was completed, the reaction solution was treated with an ultrafiltration membrane with a molecular weight cutoff of 3000 Da. The resulting filtrate was then treated with an ultrafiltration membrane with a molecular weight cutoff of 1000 Da to obtain the donkey-hide gelatin enzymatic hydrolysate reprocessing solution. S4. Preparation of donkey-hide gelatin peptide-iron chelate According to the ratio of 1 mL : 0.002 g : 0.003 g of enzymatic hydrolysis reprocessing solution of donkey-hide gelatin, ascorbic acid and ferrous chloride are added to 500 mL of enzymatic hydrolysis reprocessing solution of donkey-hide gelatin, the pH is adjusted to 5 with 10% hydrochloric acid solution, and then ferrous chloride is added. The mixture is chelated at 25℃ for 20 min, centrifuged at 4500 r / min for 5 min, and unreacted ferrous chloride is removed by nanofiltration. The nanofiltration concentrate is the donkey-hide gelatin peptide-iron chelate. S5. Preparation of donkey-hide gelatin peptide-iron chelate formulation Add genkwa extract to the donkey-hide gelatin peptide-iron chelate obtained in S4 according to the mass ratio of 1:0.01, stir and mix evenly at 75℃, and then sieve, sterilize and package.

[0028] This embodiment also provides a donkey-hide gelatin peptide-iron chelate preparation, which is prepared using the above-described preparation method.

[0029] Comparative Example 1 Comparative Example 1 provides a formulation comprising the following steps: This comparative example provides a method for preparing a formulation, comprising the following steps: S1. Preparation of donkey-hide gelatin solution According to the mass ratio of donkey-hide gelatin powder to water of 1:25, donkey-hide gelatin powder is added to water and melted at 70℃ for 20 minutes to obtain donkey-hide gelatin melted solution. After cooling the donkey-hide gelatin melted solution to room temperature, water is added to make up to 1500mL and shaken well to obtain donkey-hide gelatin stock solution. S2. Preparation of donkey-hide gelatin enzymatic hydrolysate The amount of protease added was 4% of the amount of donkey-hide gelatin powder in the donkey-hide gelatin stock solution. Trypsin was added to 1000 mL of the donkey-hide gelatin stock solution and enzymatically hydrolyzed for 4 h at a temperature of 45℃, a rotation speed of 47 r / min and a pH of 7.2. After the enzymatic hydrolysis was completed, the donkey-hide gelatin hydrolysate was treated with an ultrafiltration membrane with a molecular weight cutoff of 3000 Da. The resulting filtrate was then treated with an ultrafiltration membrane with a molecular weight cutoff of 1000 Da to obtain the donkey-hide gelatin hydrolysate. S3. Preparation of donkey-hide gelatin peptide-iron chelate The ratio of donkey-hide gelatin enzymatic hydrolysate, ascorbic acid, and ferrous chloride is 1 mL: 0.0015 g: 0.0015 g. Add ascorbic acid to 500 mL of donkey-hide gelatin enzymatic hydrolysate, adjust the pH to 4.5 with 10% hydrochloric acid solution, and then add ferrous chloride. Chelate at 22℃ for 25 min, centrifuge at 4500 r / min for 5 min, and remove unreacted ferrous chloride by nanofiltration. The nanofiltration concentrate is the donkey-hide gelatin peptide-iron chelate. S4. Preparation of donkey-hide gelatin peptide-iron chelate formulation Add genkwa extract to the donkey-hide gelatin peptide-iron chelate obtained in S3 at a mass ratio of 1:0.025, stir and mix evenly at 70℃, and then sieve, sterilize and package.

[0030] This comparative example also provides a formulation prepared using the above-described preparation method.

[0031] Comparative Example 2 The difference between this comparative example and Example 1 is that genistein is omitted in S5.

[0032] Experimental Example 1 Take 25 mg of the donkey-hide gelatin peptide-iron chelate from Examples 1-3 and Comparative Examples 1-2 respectively, add anhydrous ethanol to a concentration of 95%, and after precipitation is complete, collect the precipitates from each group, wash repeatedly with anhydrous ethanol, dissolve in 25 mL of water, and determine the Fe content. 2+ The content of [a specific ingredient] is used to calculate the chelation rate. The formula for calculating the chelation rate is as follows: Chelation rate = Fe in the precipitate 2+ Content of Fe in ferrous chloride 2+ The content was multiplied by 100%, and the results are shown in Table 1.

[0033] Table 1

[0034] As shown in Table 1, the donkey-hide gelatin peptide-iron chelate preparation obtained in this invention has a high iron content. Compared with Example 1, the iron content of Comparative Examples 1-2 is lower, indicating that the modification of donkey-hide gelatin peptide by BOC-L-cysteine-curcumin and the addition of genkwa extract both enhance the ability of donkey-hide gelatin peptide to chelate ferrous ions. Specific analysis shows that the BOC-L-cysteine-curcumin introduced into the donkey-hide gelatin peptide-iron chelate forms an O,O-bidentate six-membered ring chelate with ferrous ions through its 1,3-diketone (β-diketone) and terminal phenolic hydroxyl oxygen coordination site, thereby increasing the chelation rate of donkey-hide gelatin peptide for ferrous ions.

[0035] Experiment Example 2 The DPPH free radical scavenging activity of the formulations in Examples 1-3 and Comparative Examples 1-2 was determined by the DPPH colorimetric method. The test concentration of the formulations in Examples 1-3 and Comparative Examples 1-2 was 1 mg / mL. The results are shown in Table 2.

[0036] The superoxide anion scavenging activity of the formulations in Examples 1-3 and Comparative Examples 1-2 was determined using the pyrogallol method. The test concentration of the formulations in Examples 1-3 and Comparative Examples 1-2 was 1 mg / mL. The results are shown in Table 2.

[0037] Table 2

[0038] As shown in Table 2, the formulations obtained in Examples 1-3 of this invention have higher antioxidant capacity, while the antioxidant capacity of Comparative Example 1, which directly mixes curcumin with donkey-hide gelatin peptides, is worse. This indicates that modifying donkey-hide gelatin peptides with BOC-L-cysteine-curcumin can improve their antioxidant activity.

[0039] Experimental Example 3 3.1 Experimental Animals and Construction of Iron Deficiency Anemia Animal Model (IDA) Eighty SPF-grade ICR mice weighing 13-15g were selected and fed a 1:1 female-to-male ratio for one week of acclimatization. Ten mice were randomly selected from the control group (male-to-female ratio 1:1) and fed a standard diet (iron content 45mg Fe·kg⁻¹). -1 The remaining mice were fed a low-iron diet (iron content 10 mg Fe·kg). -1 IDA was induced by reducing iron in the basal diet. Starting from the second week of modeling, blood was collected from the tail vein of mice weekly to measure the hemoglobin (HGB) content in the blood of mice in the normal group and the model group. The modeling was considered successful when the HGB content in the mice was lower than the standard concentration of 90 g / L.

[0040] 3.2 Grouping and Administration 3.2.1 Grouping The mice that successfully developed the model were randomly divided into 6 groups of 10 mice each, with a male-to-female ratio of 1:1. These groups were Example 1-3, Comparative Example 1-2, and Model Group.

[0041] 3.2.3 Administration method Dosage: Based on the clinical daily dose of 9g of donkey-hide gelatin, and calculated according to the ratio of animal to human surface area, the equivalent dose for mice is 1.2g / kg.

[0042] Example 1 group: The preparation prepared in Example 1 was administered by gavage at a dose of 1.2 g / kg once a day for 21 consecutive days; Example 2 group: The preparation prepared in Example 2 was administered by gavage at a dose of 1.2 g / kg once a day for 21 consecutive days; Example 3 group: The preparation prepared in Example 3 was administered by gavage at a dose of 1.2 g / kg once a day for 21 consecutive days; Comparative Example 1: The preparation prepared in Comparative Example 1 was administered by gavage at a dose of 1.2 g / kg once a day for 21 consecutive days. Comparative Example 2: The preparation prepared in Comparative Example 2 was administered by gavage at a dose of 1.2 g / kg once a day for 21 consecutive days. Model group: Gavage with an equal volume of distilled water once a day for 21 consecutive days; Normal group: Gavage with an equal volume of distilled water once a day for 21 consecutive days.

[0043] 3.3 Experimental Procedure and Detection Indicators One hour after the last administration, blood was collected from the inner canthus of the eyes of mice in each group, and blood count parameters were measured using a fully automated blood cell analyzer. The blood count parameters included red blood cells (RBC), hematocrit (HCT), hemoglobin (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), directly measured mean corpuscular hemoglobin concentration (CHCM), hemoglobin distribution width (HDW), red blood cell distribution width (RDW), platelets (PLT), mean platelet volume (MPV), platelet hematocrit (PCT), and white blood cells (WBC). The results are shown in Table 3.

[0044] Table 3

[0045] As shown in Table 3, compared with the normal group, the levels of RBC, HGB, HCT, MCV, MCH, MCHC, and CHCM in the blood of the model group were significantly lower, while the levels of RDW and HDW were significantly higher, indicating that the iron deficiency anemia model was successfully established. Compared with Comparative Examples 1-2, the donkey-hide gelatin peptide-iron chelate preparations obtained in Examples 1-3 of this invention can significantly increase the levels of RBC, HGB, HCT, MCV, MCH, MCHC, and CHCM in the blood of mice with iron deficiency anemia, and decrease the levels of RDW and HDW. The above results indicate that the donkey-hide gelatin peptide-iron chelate preparations obtained in this invention have a positive effect on the treatment of iron deficiency anemia in mice.

[0046] In summary, the donkey-hide gelatin peptide of this invention is rich in active amino acid residues. Its amino group can form an amide bond with the carboxyl group of BOC-L-cysteine-curcumin through dehydration condensation, thereby enhancing the donkey-hide gelatin peptide's ability to chelate ferrous ions. The prepared donkey-hide gelatin peptide-iron chelate retains the original carboxyl and amino groups' complexing ability with ferrous ions. Furthermore, BOC-L-cysteine-curcumin forms an O,O-bidentate six-membered ring chelate with ferrous ions through its 1,3-diketone (β-diketone) and terminal phenolic hydroxyl oxygen-coordination site, increasing the chelation rate of donkey-hide gelatin peptides for ferrous ions and inhibiting the free iron-catalyzed Fenton reaction. In vitro DPPH experiments also confirmed the enhanced antioxidant activity of the complex. This process does not require the introduction of an exogenous synthetic carrier, and can simultaneously improve the water solubility of iron and the mucosal permeability of the complex system, thereby enhancing the bioavailability of iron. This provides a green, efficient, and scalable precision nutrition strategy for oral intervention in iron deficiency anemia. In addition, this invention combines the donkey-hide gelatin peptide-iron chelate with genkwa extract to form a formulation. The aromatic rings of genkwa and curcumin are stacked π-π, and their phenolic hydroxyl groups form hydrogen bonds and van der Waals force networks with the carboxyl and amino groups of the donkey-hide gelatin peptide. Spatially, the central iron ion is shielded, reducing the exchange with endogenous ligands (such as phosphate and bicarbonate), thus giving the chelate higher kinetic stability in the physiological environment.

[0047] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing a donkey-hide gelatin peptide-iron chelate preparation, characterized in that, Includes the following steps: S1. Preparation of donkey-hide gelatin solution The gelatin powder is dissolved in water, cooled, and the volume is adjusted to obtain gelatin stock solution. S2. Preparation of donkey-hide gelatin enzymatic hydrolysate Add protease to the gelatin stock solution for enzymatic hydrolysis, filter after hydrolysis to obtain gelatin hydrolysate; S3. Preparation of enzymatic hydrolysis and reprocessing solution for donkey-hide gelatin BOC-L-cysteine-curcumin, tetrabutylammonium bromide, and iodine were added to the enzymatic hydrolysate of donkey-hide gelatin. The pH was adjusted with 0.1 mol / L sodium hydroxide solution, and then the reaction was carried out. After the reaction was completed, the mixture was ultrafiltered to obtain the enzymatic hydrolysate reprocessing solution of donkey-hide gelatin. The structural formula of BOC-L-cysteine-curcumin is as follows: 2.S4. Preparation of donkey-hide gelatin peptide-iron chelate Add ascorbic acid to the enzymatic hydrolysis and reprocessing solution of donkey-hide gelatin, adjust the pH with 10% hydrochloric acid solution, then add ferrous chloride, chelate and centrifuge, and then perform nanofiltration. The nanofiltration concentrate is the donkey-hide gelatin peptide-iron chelate. S5. Preparation of donkey-hide gelatin peptide-iron chelate formulation The donkey-hide gelatin peptide-iron chelate is stirred and mixed evenly with genkwa extract, then sieved, sterilized, and packaged.

3. The method for preparing the donkey-hide gelatin peptide-iron chelate preparation according to claim 1, characterized in that, In step S1, the mass ratio of donkey-hide gelatin powder to water is 1:(20-30); the melting temperature is 60-90℃ and the time is 15-30min.

4. The preparation method of the donkey-hide gelatin peptide-iron chelate preparation according to claim 1, characterized in that, The amount of protease added in step S2 is 3-5% of the donkey-hide gelatin powder in the donkey-hide gelatin stock solution; the protease is trypsin or bromelain; the enzymatic hydrolysis conditions are: temperature 40-50℃, rotation speed 45-50r / min, pH 7-7.5; the enzymatic hydrolysis time is 3-5h.

5. The method for preparing the donkey-hide gelatin peptide-iron chelate preparation according to claim 1, characterized in that, In step S3, the amounts of BOC-L-cysteine-curcumin, tetrabutylammonium bromide, and iodine added are 8-15%, 5-12%, and 3-5% of the gelatin enzymatic hydrolysate, respectively; the pH is 9-10; and the reaction time is 3-5 hours.

6. The method for preparing the donkey-hide gelatin peptide-iron chelate preparation according to claim 4, characterized in that, The preparation method of BOC-L-cysteine-curcumin is as follows: BOC-L-cysteine, curcumin, and triethylamine are added to a tetrahydrofuran aqueous solution for reaction. After the reaction is completed, the mixture is purified to obtain BOC-L-cysteine-curcumin.

7. The method for preparing the donkey-hide gelatin peptide-iron chelate preparation according to claim 5, characterized in that, The molar ratio of curcumin, BOC-L-cysteine, and triethylamine is 1:(1.2-1.6):(1.2-1.6); the volume ratio of tetrahydrofuran to water in the tetrahydrofuran aqueous solution is 1:5; the reaction temperature is 20-25℃, and the reaction time is 3-5h.

8. The method for preparing the donkey-hide gelatin peptide-iron chelate preparation according to claim 1, characterized in that, In step S4, the ratio of the amount of donkey-hide gelatin enzymatic hydrolysis reprocessing solution, ascorbic acid, and ferrous chloride is 1 mL: (0.001-0.002) g: (0.003-0.006) g; the pH is adjusted to 4-5; the chelation temperature is 20-25℃ and the time is 20-30 min.

9. The method for preparing the donkey-hide gelatin peptide-iron chelate preparation according to claim 1, characterized in that, In step S5, the mass ratio of the donkey-hide gelatin peptide-iron chelate to genkwa extract is (10-20):(0.1-0.5); the stirring temperature is 65-75℃.

10. A donkey-hide gelatin peptide-iron chelate preparation, characterized in that, It was prepared using the preparation method of the donkey-hide gelatin peptide-iron chelate preparation according to any one of claims 1-8.

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