A chelated iron e-jiao sugar peptide composition and a preparation method thereof

Abstract

The application belongs to the technical field of health food, and particularly relates to a chelated iron donkey-hide gelatin sugar peptide composition and a preparation method thereof. The preparation method comprises the following steps: adding a protease to a gel solution for enzymolysis, performing ultrafiltration grading after the enzymolysis is completed, collecting a concentrated solution, and obtaining a donkey-hide gelatin peptide enzymolysis solution; adding an ethanol solution of a ribose derivative to the donkey-hide gelatin peptide enzymolysis solution for a Maillard reaction, adjusting the pH after the reaction is completed, and obtaining a donkey-hide gelatin peptide solution; adding a ferrous chloride solution to the donkey-hide gelatin peptide solution for a stirring reaction, removing unreacted ferrous chloride by nanofiltration treatment after the reaction is completed, and obtaining modified chelated iron donkey-hide gelatin sugar peptide powder after freeze-drying of the nanofiltration concentrated solution; adding beta-cyclodextrin to a phosphate buffer solution, adding the modified chelated iron donkey-hide gelatin sugar peptide powder and vitamin C, and obtaining the chelated iron donkey-hide gelatin sugar peptide composition through ultrasonic treatment, freeze-drying and grinding. The composition has a high iron content.

Description

Technical Field

[0001] This invention belongs to the field of health food technology, and in particular relates to a chelated iron-containing donkey-hide gelatin glycopeptide composition and its preparation method. Background Technology

[0002] Peptide-iron chelates are metal-chelated peptides composed of bioactive peptides and ferrous ions (Fe). 2+ It is formed by chelation. These complexes can act as Fe in the body. 2+ It is an effective carrier for iron, promoting the absorption and transport of iron, and has a positive effect on improving related diseases caused by iron deficiency (such as iron deficiency anemia).

[0003] Donkey-hide gelatin, a traditional tonic food with both medicinal and edible properties, has shown good effects in improving anemia symptoms and enhancing immune system function. The process involves degrading donkey-hide gelatin into donkey-hide gelatin peptides, reacting them with reducing sugars via a Maillard reaction to obtain donkey-hide gelatin glycopeptides, and then reacting with Fe... 2+ Chelation yields "chelated iron-fortified donkey-hide gelatin glycopeptides," which exhibit high absorption rates and low irritation, making them a promising new iron supplement. However, the practical application of this chelate currently faces several challenges, primarily including low iron ion chelation rates, poor complex stability, and the hygroscopic nature and poor storage properties of the donkey-hide gelatin peptides themselves. Furthermore, the product often exhibits unpleasant flavors (such as a rusty taste), affecting its acceptability. Although ferrous ions are physiologically superior to ferric ions, their chemical instability and susceptibility to oxidation further limit the actual effectiveness of this iron supplement. Therefore, formulation optimization is urgently needed to improve the overall performance of chelated iron-fortified donkey-hide gelatin glycopeptides. Summary of the Invention

[0004] 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 chelated iron-containing glycopeptide composition.

[0005] The second objective of this invention is to provide a chelated iron-containing glycopeptide composition with high iron content and stability.

[0006] One of the objectives of this invention is achieved through the following technical solution: A method for preparing a chelated iron-containing donkey-hide gelatin glycopeptide composition includes the following steps: (1) Add protease to the gelatin solution for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, perform ultrafiltration fractionation, collect the concentrated solution, and obtain the gelatin peptide hydrolysate. (2) Add an ethanol solution of ribose derivative to the enzymatic hydrolysate of donkey-hide gelatin peptide to carry out the Maillard reaction. After the reaction is completed, adjust the pH to obtain the donkey-hide gelatin peptide solution. The structural formula of the ribose derivative is as follows: (3) Add ferrous chloride solution to the donkey-hide gelatin peptide solution and stir to react. After the reaction is completed, remove unreacted ferrous chloride by nanofiltration. After freeze-drying the nanofiltration concentrate, obtain modified chelated iron donkey-hide gelatin glycopeptide powder. (4) Add β-cyclodextrin to phosphate buffer, then add the modified chelated iron-fortified glycopeptide powder and vitamin C, and sonicate, freeze dry and grind to obtain the chelated iron-fortified glycopeptide composition.

[0007] Furthermore, the ultrafiltration fractionation in step (1) is as follows: the enzymatic hydrolysate is first treated with an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, and the resulting filtrate is then treated with an ultrafiltration membrane with a molecular weight cutoff of 1000 Da.

[0008] Further, the enzymatic hydrolysis conditions in step (1) are: pH 5-6.5, temperature 50-60℃, and hydrolysis time 3-4h; the amount of protease added is 0.1-0.5% of the mass of the gelatin solution; the protease is composed of papain and bromelain in a mass ratio of 1:(1-2); the preparation method of the gelatin solution is: add 2-3 times the amount of water to the gelatin block to melt and boil, keep warm for 15-25min, then add 1-1.5 times the amount of water of the gelatin, and homogenize 2-3 times at 55-70℃ and 125-140MPa.

[0009] Furthermore, in step (2), the amount of ribose derivative ethanol solution added is 0.1-0.5% of the mass of the donkey-hide gelatin peptide hydrolysate; the concentration of ribose derivative in the ribose derivative ethanol solution is 40-80 mg / g; the Maillard reaction conditions are: pH 10-11, temperature 80-90℃, and reaction time 2-3 h; the pH is adjusted to 7-7.2.

[0010] Furthermore, the method for preparing the ribose derivative is as follows: a. Add cypermethrin to anhydrous pyridine, then add methanesulfonyl chloride and stir overnight. Concentrate under vacuum to dryness, then redissolve the residue in N,N-dimethylformamide, add sodium azide, and react at 60-65℃ for 2-6 hours. After the reaction is complete, purify to obtain intermediate 1. The structural formula of intermediate 1 is as follows: b. Add the intermediate 1 and acetylated ribose to a mixture of tetrahydrofuran / water / tert-butanol, add copper sulfate pentahydrate and sodium ascorbate, react overnight at 25-30°C, purify after the reaction is complete to obtain the ribose derivative; The structural formula of the acetylated ribose is as follows: .

[0011] Furthermore, in step a, the ratio of oxaliplatin, methanesulfonyl chloride, and sodium azide is 1 mg: (0.16-0.19) μL: (0.27-0.3) mg; in step b, the molar ratio of intermediate 1, acetylated ribose, copper sulfate pentahydrate, and sodium ascorbate is 1: (1-1.25): (0.2-0.25): (0.4-0.5); and the volume ratio of tetrahydrofuran, water, and tert-butanol in the tetrahydrofuran / water / tert-butanol mixture is 3:1:1.

[0012] Furthermore, the preparation method of the acetylated ribose is as follows: Linoleic acid was added to dichloromethane, followed by 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. After stirring for 30-45 min, (S)-3-amino-5-hexanoate was added and the reaction was allowed to proceed overnight. After the reaction was completed, the mixture was purified to obtain intermediate 2. The structural formula of intermediate 2 is as follows: The intermediate 2,1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added to N,N-dimethylformamide, followed by the addition of 4-dimethylaminopyridine. After stirring and activating for 30-45 min, ribose was added and reacted for 12-24 h. The reaction was terminated by adding water. After purification of the reaction solution, the acetylated ribose was obtained.

[0013] Further steps The molar ratio of linoleic acid, (S)-3-amino-5-hexanoate, 4-dimethylaminopyridine, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:(1-1.1):(1.3-1.7):(1.3-1.7); Step The molar ratio of intermediate 2, ribose, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine is 1:(1-1.1):(1.5-2):(1.5-2).

[0014] Furthermore, in step (3), the amount of ferrous chloride solution added is 1-5% of the mass of the donkey-hide gelatin peptide solution; the concentration of the ferrous chloride solution is 1-1.5 mol / L; and the stirring reaction time is 30-45 min.

[0015] Furthermore, in step (4), the mass ratio of the modified chelated iron-containing glycopeptide powder, β-cyclodextrin, and vitamin C is 1:(18-20):(0.1-0.2); the temperature of the ultrasound is 40-60℃, and the time is 20-30 min.

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

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention first modifies the chelated iron-containing donkey-hide gelatin glycopeptide. Specifically, intermediate 2 is prepared by amidation reaction of linoleic acid and (S)-3-amino-5-hexanoate, followed by esterification reaction with ribose to obtain acetylated ribose. The acetylated ribose is then reacted with azido-containing arbutin via a click reaction to synthesize a ribose derivative containing both arbutin and linoleic acid fragments. This derivative undergoes a Maillard reaction with a donkey-hide gelatin peptide solution, followed by chelation with ferrous ions to obtain the modified chelated iron-containing donkey-hide gelatin glycopeptide. Subsequently, the modified chelated iron-containing donkey-hide gelatin glycopeptide and vitamin C are encapsulated with β-cyclodextrin to obtain a chelated iron-containing donkey-hide gelatin glycopeptide composition. After the above modification treatment, arbutin and linoleic acid are successfully introduced and form a multi-nitrogen heterocyclic structure. Harmonyside, rich in phenolic hydroxyl groups, can synergistically interact with the polycyclic nitrogen ring to effectively enhance its chelating ability for ferrous ions. Simultaneously, harmonyside itself has a sweet taste, which can mask the rusty taste of ferrous ions and improve the product's flavor. On the other hand, the introduction of linoleic acid enhances the hydrophobicity of the donkey-hide gelatin glycopeptide, not only helping to reduce its hygroscopicity but also allowing it to enter the hydrophobic cavity of β-cyclodextrin through hydrophobic interactions, thereby improving the encapsulation efficiency of subsequent microencapsulation and synergistically enhancing overall storage stability. Furthermore, linoleic acid possesses antioxidant properties, which can inhibit Fe... 2+ It catalyzes lipid peroxidation and promotes transmembrane transport of iron, thereby enhancing the stability of ferrous ions. Furthermore, the vitamin C contained in the composition possesses strong reducing power, effectively maintaining the stable state of easily oxidized ferrous ions, thus improving their bioavailability and absorption rate in the body. Detailed Implementation

[0018] 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.

[0019] Preparation Example 1 Preparation Example 1 provides an acetylated ribose, the preparation method of which includes the following steps: Following a reaction ratio of 1 mmol:1 mmol:1.5 mmol:1.5 mmol:1.5 mmol:12 mL, linoleic acid was added to dichloromethane, followed by 4-dimethylaminopyridine (DMAP) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI). After stirring for 40 min, (S)-3-amino-5-hexanoate (CAS: 270596-46-8) was added, and the mixture was reacted overnight at room temperature. After the reaction was complete, the mixture was diluted with dichloromethane, washed successively with water and saturated brine, dried over anhydrous MgSO4, evaporated under reduced pressure, and purified by column chromatography to obtain intermediate 2. The NMR and mass spectrometry results of intermediate 2 are as follows: 1 HNMR (C 24 H 39 O3N, 400MHz, DMSO-d6) δ: 0.86-0.90 (m, 3H), 1.24-1.35 (m, 14H), 1.51-1.55 (m, 2H), 2.03-2.12 (m, 3H), 2.14-2.18 (m, 4H), 2.29-2.37 (m, 2H), 2.54-2.58 (m, 1H), 2.78-2.82 (t, 2H), 2.91-2.95 (t, 1H), 3.88-3.92 (m , 1H), 5.27-5.31 (m, 2H), 5.41-5.45 (m, 2H), 8.14 (s, 1H), 12.01 (s, 1H). MS(ESI) m / z=389.29[M].

[0020] According to the ratio of intermediate 2, ribose, EDCI, DMAP, and N,N-dimethylformamide (1 mmol: 1 mmol: 1.6 mmol: 1.6 mmol: 3.5 mL), intermediate 2 and EDCI were added to N,N-dimethylformamide, followed by DMAP. After stirring and activating for 40 min, ribose was added and the reaction was allowed to proceed for 16 h. The reaction was terminated by adding water. The reaction solution was extracted with dichloromethane, washed with saturated brine, dried over anhydrous MgSO4, filtered, evaporated under reduced pressure, and purified by column chromatography to obtain the acetylated ribose. The NMR and mass spectrometry results of the acetylated ribose are as follows: 1 HNMR (C 29 H 47O7N, 400MHz, DMSO-d6) δ: 0.86-0.90 (m, 3H), 1.24-1.35 (m, 14H), 1.51-1.55 (m, 2H), 2.03- 2.12 (m, 3H), 2.14-2.18 (m, 4H), 2.31-2.37 (m, 2H), 2.56-2.60 (m, 1H), 2.78-2.82 (t, 2H), 2.91-2.95 (t, 1H), 3.70-3.74 (m, 1H), 4.06-4.11 (m, 2H), 4.18-4.22 (m, 1H), 4.31-4.39 (m , 4H), 5.18 (s, 1H), 5.27-5.31 (m, 2H), 5.41-5.45 (m, 2H), 8.14 (s, 1H), 9.70-9.74 (d, 1H). MS (ESI)m / z=521.34[M].

[0021] Preparation Example 2 Preparation Example 2 provides an acetylated ribose, the preparation method of which includes the following steps: Following the ratio of linoleic acid, (S)-3-amino-5-hexanoate, DMAP, EDCI, and dichloromethane of 1 mmol: 1 mmol: 1.3 mmol: 1.3 mmol: 10 mL, linoleic acid was added to dichloromethane, followed by DMAP and EDCI. After stirring for 30 min, (S)-3-amino-5-hexanoate was added, and the mixture was reacted overnight at room temperature. After the reaction was complete, dichloromethane was added for dilution, followed by washing with water and saturated brine, drying with anhydrous MgSO4, evaporating under reduced pressure, and purifying by column chromatography to obtain intermediate 2. The NMR and mass spectrometry results of intermediate 2 were the same as those in preparation example 1.

[0022] According to the ratio of intermediate 2, ribose, EDCI, DMAP, and N,N-dimethylformamide of 1 mmol: 1 mmol: 1.5 mmol: 1.5 mmol: 3 mL, intermediate 2 and EDCI were added to N,N-dimethylformamide, followed by DMAP. After stirring and activating for 30 min, ribose was added and the reaction was carried out for 24 h. The reaction was terminated by adding water. The reaction solution was extracted with dichloromethane, washed with saturated brine, dried over anhydrous MgSO4, filtered, evaporated under reduced pressure, and purified by column chromatography to obtain the acetylated ribose. The NMR and mass spectrometry results of the acetylated ribose were the same as those in Preparation Example 1.

[0023] Preparation Example 3 Preparation Example 3 provides an acetylated ribose, the preparation method of which includes the following steps: Following the ratio of linoleic acid, (S)-3-amino-5-hexanoate, DMAP, EDCI, and dichloromethane of 1 mmol: 1.1 mmol: 1.7 mmol: 1.7 mmol: 13 mL, linoleic acid was added to dichloromethane, followed by DMAP and EDCI. After stirring for 45 min, (S)-3-amino-5-hexanoate was added, and the mixture was reacted overnight at room temperature. After the reaction was complete, dichloromethane was added for dilution, followed by washing with water and saturated brine, drying with anhydrous MgSO4, evaporating under reduced pressure, and purifying by column chromatography to obtain intermediate 2. The NMR and mass spectrometry results of intermediate 2 were the same as those in preparation example 1.

[0024] According to the ratio of intermediate 2, ribose, EDCI, DMAP, and N,N-dimethylformamide (1 mmol: 1.1 mmol: 2 mmol: 2 mmol: 4 mL), intermediate 2 and EDCI were added to N,N-dimethylformamide, followed by DMAP. After stirring and activating for 45 min, ribose was added and the reaction was allowed to proceed for 12 h. The reaction was terminated by adding water. The reaction solution was extracted with dichloromethane, washed with saturated brine, dried over anhydrous MgSO4, filtered, evaporated under reduced pressure, and purified by column chromatography to obtain the acetylated ribose. The NMR and mass spectrometry results of the acetylated ribose were the same as those in Preparation Example 1.

[0025] Preparation Example 4 Preparation Example 4 provides a ribose derivative, the preparation method of which includes the following steps: a. Following the ratio of harvestingin, methanesulfonyl chloride, sodium azide, anhydrous pyridine, and N,N-dimethylformamide of 1 mg:0.18 μL:0.28 mg:30 μL:50 μL, harvestingin was added to anhydrous pyridine, followed by slow addition of methanesulfonyl chloride. The mixture was stirred overnight at room temperature and concentrated to dryness under vacuum. The residue was then redissolved in N,N-dimethylformamide, and sodium azide was added. The mixture was reacted at 62 °C for 4 h. After the reaction was complete, column chromatography was used to purify the intermediate to obtain intermediate 1. The NMR and mass spectrometry results of intermediate 1 are as follows: 1 HNMR (C 21 H 19 O 10N3, 400MHz, DMSO-d6) δ: 1.1-1.4 (m, 1H), 1.5-1.8 (m, 1H), 3.58-3.62 (m, 2H), 3.68-3.72 (m, 1H), 4.06-4.10 (m, 1H), 4.37 (s, 1H), 4.51 (s, 2H) ), 4.86-4.90 (d, 1H), 6.05 (s, 1H), 6.52 (s, 1H), 6.71 (s, 1H), 6.80-6. 84 (d, 1H), 7.02-7.06 (d, 1H), 9.48 (s, 2H), 9.68 (s, 1H), 16.47 (s, 1H). MS(ESI)m / z=473.11[M].

[0026] b. Following the ratio of intermediate 1, acetylated ribose, copper sulfate pentahydrate, sodium ascorbate, and tetrahydrofuran / water / tert-butanol mixture (1 μmol: 1.1 μmol: 0.22 μmol: 0.45 μmol: 27 μL), intermediate 1 and acetylated ribose were added to the tetrahydrofuran / water / tert-butanol mixture (v / v / v, 3:1:1). Copper sulfate pentahydrate and sodium ascorbate were then added, and the mixture was reacted overnight at 28°C. After the reaction was complete, the mixture was extracted with ethyl acetate / water, dried over anhydrous magnesium sulfate, filtered, concentrated by rotary evaporation, and purified by column chromatography to obtain the ribose derivative. The NMR and mass spectrometry results of the ribose derivative are as follows: 1 HNMR (C 50 H 66 O 17N4, 400MHz, DMSO-d6) δ: 0.86-0.90 (m, 3H), 1.24-1.35 (m, 14H), 1.51-1.55 (m, 2H), 2.02-2.07 (m, 2H), 2.14-2.18 (m, 4H), 2.31-2.35 (m, 1H), 2.56-2.65 (m, 2H), 2.78-2.82 (m, 2H), 2.86-2.90 (m, 1H), 3.58-3.62 ( m, 2H), 3.68-3.74 (m, 3H), 3.94-3.98 (m, 1H), 4.06-4.11 (m, 3H), 4.31- 4.39 (m, 5H), 4.51 (s, 2H), 4.59-4.63 (m, 1H), 4.86-4.90 (d, 1H), 5.18 (s, 1H), 5.27-5.31 (m, 2H), 5.41-5.45 (m, 2H), 6.05 (s, 1H), 6.52 (s, 1H ), 6.71 (s, 1H), 6.80-6.84 (d, 1H), 7.02-7.06 (d, 1H), 7.59 (s, 1H), 8. 14 (s, 1H), 9.48 (s, 2H), 9.68 (s, 1H), 9.70-9.74 (d, 1H), 16.47 (s, 1H). MS(ESI) m / z=994.44[M].

[0027] Preparation Example 5 Preparation Example 5 provides a ribose derivative, the preparation method of which includes the following steps: a. Following the ratio of cypermethrin, methanesulfonyl chloride, sodium azide, anhydrous pyridine, and N,N-dimethylformamide of 1 mg: 0.16 μL: 0.27 mg: 20 μL: 40 μL, cypermethrin was added to anhydrous pyridine, followed by slow addition of methanesulfonyl chloride. The mixture was stirred overnight at room temperature and concentrated to dryness under vacuum. The residue was then redissolved in N,N-dimethylformamide, and sodium azide was added. The mixture was reacted at 60 °C for 6 h. After the reaction was complete, the mixture was purified by column chromatography to obtain intermediate 1. The NMR and mass spectrometry results of intermediate 1 were the same as those in preparation example 4.

[0028] b. According to the ratio of intermediate 1, acetylated ribose, copper sulfate pentahydrate, sodium ascorbate, and tetrahydrofuran / water / tert-butanol mixture 1 μmol: 1 μmol: 0.2 μmol: 0.4 μmol: 25 μL, intermediate 1 and acetylated ribose were added to the tetrahydrofuran / water / tert-butanol mixture (v / v / v, 3:1:1), copper sulfate pentahydrate and sodium ascorbate were added, and the mixture was reacted overnight at 25°C. After the reaction was completed, the mixture was extracted with ethyl acetate / water, dried over anhydrous magnesium sulfate, filtered, concentrated by rotary evaporation, and purified by column chromatography to obtain the ribose derivative. The NMR and mass spectrometry results of the ribose derivative were the same as those in Preparation Example 4.

[0029] Preparation Example 6 Preparation Example 6 provides a ribose derivative, the preparation method of which includes the following steps: a. Following the ratio of cypermethrin, methanesulfonyl chloride, sodium azide, anhydrous pyridine, and N,N-dimethylformamide of 1 mg: 0.19 μL: 0.3 mg: 40 μL: 70 μL, cypermethrin was added to anhydrous pyridine, followed by the slow addition of methanesulfonyl chloride. The mixture was stirred overnight at room temperature and concentrated to dryness under vacuum. The residue was then redissolved in N,N-dimethylformamide, and sodium azide was added. The mixture was reacted at 65 °C for 2 h. After the reaction was complete, the mixture was purified by column chromatography to obtain intermediate 1. The NMR and mass spectrometry results of intermediate 1 were the same as those in preparation example 4.

[0030] b. According to the ratio of intermediate 1, acetylated ribose, copper sulfate pentahydrate, sodium ascorbate, and tetrahydrofuran / water / tert-butanol mixture 1 μmol: 1.25 μmol: 0.25 μmol: 0.5 μmol: 30 μL, intermediate 1 and acetylated ribose were added to the tetrahydrofuran / water / tert-butanol mixture (v / v / v, 3:1:1), copper sulfate pentahydrate and sodium ascorbate were added, and the mixture was reacted overnight at 30°C. After the reaction was completed, the mixture was extracted with ethyl acetate / water, dried over anhydrous magnesium sulfate, filtered, concentrated by rotary evaporation, and purified by column chromatography to obtain the ribose derivative. The NMR and mass spectrometry results of the ribose derivative were the same as those in Preparation Example 4.

[0031] Preparation Example 7 The difference between Preparation Example 7 and Preparation Example 4 is that ribose is used instead of acetylated ribose, otherwise the same as Preparation Example 4.

[0032] Example 1 Example 1 provides a method for preparing a chelated iron-containing donkey-hide gelatin glycopeptide composition, comprising the following steps: (1) Add 2.5 times the amount of water to the donkey-hide gelatin block, melt, boil, keep warm for 20 minutes, and then add 1.2 times the amount of water of the donkey-hide gelatin. Homogenize twice at 60℃ and 130MPa to obtain the gel solution. Add the protease to the gel solution at a rate of 0.3% of the mass of the gel solution. The protease is composed of papain and bromelain in a mass ratio of 1:1.5. Under the conditions of pH 6 and temperature of 55℃, the enzymatic hydrolysis time is 3.5h. After the enzymatic hydrolysis is completed, the enzymatic hydrolysate is first treated with an ultrafiltration membrane with a molecular weight cutoff of 3000Da. The obtained filtrate is then treated with an ultrafiltration membrane with a molecular weight cutoff of 1000Da. Collect the concentrated liquid to obtain the donkey-hide gelatin peptide enzymatic hydrolysate. (2) The amount of ethanol solution of ribose derivative added is 0.3% of the mass of the donkey-hide gelatin peptide hydrolysate. The ethanol solution of ribose derivative of Preparation Example 4 (the concentration of ribose derivative is 60 mg / g) is added to the above donkey-hide gelatin peptide hydrolysate. The Maillard reaction is carried out for 2.5 h at pH 10.5 and temperature 85 °C. After the reaction is completed, the pH is adjusted to 7.1 to obtain the donkey-hide gelatin peptide solution. (3) The amount of ferrous chloride solution added is 5% of the mass of the donkey-hide gelatin peptide solution. A ferrous chloride solution with a concentration of 1.3 mol / L is added to the donkey-hide gelatin peptide solution and stirred for 40 min. After the reaction is completed, nanofiltration is used to remove unreacted ferrous chloride. The nanofiltration concentrate is freeze-dried to obtain modified chelated iron donkey-hide gelatin peptide powder. (4) According to the mass ratio of modified chelated iron-fortified glycopeptide powder, β-cyclodextrin and vitamin C of 1:19:0.15, β-cyclodextrin was added to phosphate buffer, and then the modified chelated iron-fortified glycopeptide powder and vitamin C were added. The mixture was sonicated at 50°C for 25 min, freeze-dried and ground to obtain the chelated iron-fortified glycopeptide composition.

[0033] Example 1 also provides a chelated iron-containing glycopeptide composition, which is prepared by the above method.

[0034] Example 2 Example 2 provides a method for preparing a chelated iron-containing donkey-hide gelatin glycopeptide composition, comprising the following steps: (1) Add twice the amount of water to the donkey-hide gelatin block, melt, boil, keep warm for 15 minutes, then add water equal to the weight of the donkey-hide gelatin, homogenize three times at 55℃ and 125MPa to obtain the gel solution; add protease to the gel solution at a ratio of 0.1% of the weight of the gel solution. The protease is composed of papain and bromelain in a mass ratio of 1:1. Under the conditions of pH 5 and temperature of 50℃, the enzymatic hydrolysis time is 3h. After the enzymatic hydrolysis is completed, the enzymatic hydrolysate is first treated with an ultrafiltration membrane with a molecular weight cutoff of 3000Da, and the obtained filtrate is then treated with an ultrafiltration membrane with a molecular weight cutoff of 1000Da. Collect the concentrated liquid to obtain the donkey-hide gelatin peptide enzymatic hydrolysate. (2) The amount of ethanol solution of ribose derivative added is 0.1% of the mass of the donkey-hide gelatin peptide hydrolysate. The ethanol solution of ribose derivative of Preparation Example 5 (the concentration of ribose derivative is 40 mg / g) is added to the above donkey-hide gelatin peptide hydrolysate. The Maillard reaction is carried out for 3 h at pH 10 and temperature 80 °C. After the reaction is completed, the pH is adjusted to 7 to obtain the donkey-hide gelatin peptide solution. (3) The amount of ferrous chloride solution added is 3% of the mass of the donkey-hide gelatin peptide solution. A ferrous chloride solution with a concentration of 1 mol / L is added to the donkey-hide gelatin peptide solution and stirred for 30 min. After the reaction is completed, nanofiltration is used to remove unreacted ferrous chloride. The nanofiltration concentrate is freeze-dried to obtain modified chelated iron donkey-hide gelatin peptide powder. (4) According to the mass ratio of modified chelated iron-fortified glycopeptide powder, β-cyclodextrin and vitamin C 1:18:0.1, β-cyclodextrin was added to phosphate buffer, and then the modified chelated iron-fortified glycopeptide powder and vitamin C were added. The mixture was sonicated at 40°C for 30 min, freeze-dried and ground to obtain the chelated iron-fortified glycopeptide composition.

[0035] Example 2 also provides a chelated iron-containing glycopeptide composition, which is prepared by the above method.

[0036] Example 3 Example 3 provides a method for preparing a chelated iron-containing donkey-hide gelatin glycopeptide composition, comprising the following steps: (1) Add 3 times the amount of water to the donkey-hide gelatin block, melt, boil, keep warm for 25 minutes, and then add 1.5 times the amount of water of the donkey-hide gelatin. Homogenize twice at 70℃ and 140MPa to obtain the gel solution. Add the protease to the gel solution at a rate of 0.5% of the mass of the gel solution. The protease is composed of papain and bromelain in a mass ratio of 1:2. Under the conditions of pH 6.5 and temperature 60℃, the enzymatic hydrolysis time is 3h. After the enzymatic hydrolysis is completed, the enzymatic hydrolysate is first treated with an ultrafiltration membrane with a molecular weight cutoff of 3000Da. The obtained filtrate is then treated with an ultrafiltration membrane with a molecular weight cutoff of 1000Da. Collect the concentrated liquid to obtain the donkey-hide gelatin peptide enzymatic hydrolysate. (2) The amount of ethanol solution of ribose derivative added is 0.5% of the mass of the donkey-hide gelatin peptide hydrolysate. The ethanol solution of ribose derivative of Preparation Example 6 (the concentration of ribose derivative is 80 mg / g) is added to the above donkey-hide gelatin peptide hydrolysate. The Maillard reaction is carried out for 2 h at pH 11 and temperature 90 °C. After the reaction is completed, the pH is adjusted to 7 to obtain the donkey-hide gelatin peptide solution. (3) The amount of ferrous chloride solution added is 1% of the mass of the donkey-hide gelatin peptide solution. A ferrous chloride solution with a concentration of 1.5 mol / L is added to the donkey-hide gelatin peptide solution and stirred for 45 min. After the reaction is completed, nanofiltration is used to remove unreacted ferrous chloride. The nanofiltration concentrate is freeze-dried to obtain modified chelated iron donkey-hide gelatin peptide powder. (4) According to the mass ratio of modified chelated iron-fortified glycopeptide powder, β-cyclodextrin and vitamin C 1:20:0.2, β-cyclodextrin was added to phosphate buffer, and then the modified chelated iron-fortified glycopeptide powder and vitamin C were added. The mixture was sonicated at 60°C for 20 min, freeze-dried and ground to obtain the chelated iron-fortified glycopeptide composition.

[0037] Example 3 also provides a chelated iron-containing glycopeptide composition, which is prepared by the above method.

[0038] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the acetylated ribose of Preparation Example 1 was used to replace the ribose derivative of Preparation Example 4, and the rest was the same as in Example 1.

[0039] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that the compound of Preparation Example 7 was used to replace the ribose derivative of Preparation Example 4, and the rest is the same as in Example 1.

[0040] Experimental Example 1 Take 2g of the compositions 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 25mL of water, and determine their 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.

[0041] Table 1

[0042] As shown in Table 1, the chelated iron-containing donkey-hide gelatin peptide composition obtained in this invention has a high iron content.

[0043] Experiment Example 2 Hygroscopicity: 2g of the compositions of Examples 1-3 and Comparative Examples 1-2 were taken and placed in a desiccator with a relative humidity of 90%±5%. The desiccator was placed in an incubator with a set temperature of 25°C. Samples were taken on the 5th and 10th days to determine the hygroscopicity of each composition. The results are shown in Table 2.

[0044] Changes in total iron and total peptides: 2g of the compositions from Examples 1-3 and Comparative Examples 1-2 were packaged in aluminum foil composite film and placed in a desiccator at 40℃±2℃ and 75%±5% relative humidity for 3 months. Samples were taken at the end of 0, 1, 2, and 3 months. Following the guidelines for stability testing of raw materials and preparations under General Chapter 9001 of the 2020 edition of the Chinese Pharmacopoeia, the total iron content and total peptide content were determined, and the changes in total iron and total peptides compared to 0 months were calculated. The changes in total iron and total peptides were used to measure the stability of the chelated iron-fortified donkey-hide gelatin peptide composition under long-term experimental conditions. The results are shown in Table 3.

[0045] Table 2

[0046] Table 3

[0047] As shown in Tables 2-3, compared with Comparative Examples 1-2, the chelated iron-containing donkey-hide gelatin peptide composition obtained in this invention has lower hygroscopicity and higher storage stability. This is because the present invention introduces hydrophobic linoleic acid segments into the chelate, which enhances the hydrophobicity of the donkey-hide gelatin peptide, thereby reducing its hygroscopicity and enhancing the storage stability of the chelate.

[0048] Experimental Example 3 Take 2g of each group of chelated iron-fortified donkey-hide gelatin peptide composition and modified chelated iron-fortified donkey-hide gelatin peptide powder, and use Fe... 2+ The content is used as an indicator, and the encapsulation rate is calculated according to the following formula: Encapsulation rate = Fe in the chelated iron-containing donkey-hide gelatin peptide composition 2+ Fe content in modified chelated iron-containing donkey-hide gelatin peptide powder 2+ The content of [the substance] is shown in Table 4.

[0049] Table 4

[0050] As shown in Table 4, compared with Comparative Examples 1-2, the composition obtained by the present invention has a higher encapsulation efficiency. This is because the present invention introduces hydrophobic linoleic acid segments into the chelate, which enhances the hydrophobicity of the donkey-hide gelatin glycopeptide, allowing the donkey-hide gelatin glycopeptide to enter the hydrophobic cavity of β-cyclodextrin through hydrophobic interactions, thereby improving the encapsulation efficiency of subsequent microencapsulation and synergistically enhancing the overall storage stability.

[0051] In summary, this invention first modifies the chelated iron-containing donkey-hide gelatin glycopeptide. Specifically, intermediate 2 is prepared by amidation reaction of linoleic acid and (S)-3-amino-5-hexanoate, followed by esterification reaction with ribose to obtain acetylated ribose. The acetylated ribose is then reacted with azido-containing arbutin via a click reaction to synthesize a ribose derivative containing both arbutin and linoleic acid fragments. This derivative undergoes a Maillard reaction with the donkey-hide gelatin peptide solution, followed by chelation with ferrous ions to obtain the modified chelated iron-containing donkey-hide gelatin glycopeptide. Subsequently, the modified chelated iron-containing donkey-hide gelatin glycopeptide and vitamin C are encapsulated using β-cyclodextrin to obtain the chelated iron-containing donkey-hide gelatin glycopeptide composition. After the above modification treatment, arbutin and linoleic acid are successfully introduced, forming a multi-nitrogen heterocyclic structure. Harmonyside, rich in phenolic hydroxyl groups, can synergistically interact with the polycyclic nitrogen ring to effectively enhance its chelating ability for ferrous ions. Simultaneously, harmonyside itself has a sweet taste, which can mask the rusty taste of ferrous ions and improve the product's flavor. On the other hand, the introduction of linoleic acid enhances the hydrophobicity of the donkey-hide gelatin glycopeptide, not only helping to reduce its hygroscopicity but also allowing it to enter the hydrophobic cavity of β-cyclodextrin through hydrophobic interactions, thereby improving the encapsulation efficiency of subsequent microencapsulation and synergistically enhancing overall storage stability. Furthermore, linoleic acid possesses antioxidant properties, which can inhibit Fe... 2+ It catalyzes lipid peroxidation and promotes transmembrane transport of iron, thereby enhancing the stability of ferrous ions. Furthermore, the vitamin C contained in the composition possesses strong reducing power, effectively maintaining the stable state of easily oxidized ferrous ions, thus improving their bioavailability and absorption rate in the body.

[0052] Experiment Example 4 4.1 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.

[0053] 4.2 Grouping and Administration 4.2.1 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.

[0054] 4.2.2 Administration method Dosage: Based on the clinical daily dose of 9g of donkey-hide gelatin, and converted according to the ratio of animal to human surface area, the equivalent dose for mice is 1.5g / kg.

[0055] Example 1 group: The composition prepared in Example 1 was administered by gavage at a dose of 1.5 g / kg once a day for 21 consecutive days; Example 2 group: The composition prepared in Example 2 was administered by gavage at a dose of 1.5 g / kg once a day for 21 consecutive days; Example 3 group: The composition prepared in Example 3 was administered by gavage at a dose of 1.5 g / kg once a day for 21 consecutive days; Comparative Example 1: The composition prepared in Comparative Example 1 was administered by gavage at a dose of 1.5 g / kg once a day for 21 consecutive days. Comparative Example 2: The composition prepared in Comparative Example 2 was administered by gavage at a dose of 1.5 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.

[0056] 4.3 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 5.

[0057] Table 5

[0058] As shown in Table 5, 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 compositions 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 demonstrate that the compositions obtained in this invention have a positive effect on the treatment of iron deficiency anemia in mice.

[0059] 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 chelated iron-containing donkey-hide gelatin glycopeptide composition, characterized in that, Includes the following steps: (1) Add protease to the gelatin solution for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, perform ultrafiltration fractionation, collect the concentrated solution, and obtain the gelatin peptide hydrolysate. (2) Add an ethanol solution of ribose derivative to the enzymatic hydrolysate of donkey-hide gelatin peptide to carry out the Maillard reaction. After the reaction is completed, adjust the pH to obtain the donkey-hide gelatin peptide solution. The structural formula of the ribose derivative is as follows: (3) Add ferrous chloride solution to the donkey-hide gelatin peptide solution and stir to react. After the reaction is completed, remove unreacted ferrous chloride by nanofiltration. After freeze-drying the nanofiltration concentrate, obtain modified chelated iron donkey-hide gelatin glycopeptide powder. (4) Add β-cyclodextrin to phosphate buffer, then add the modified chelated iron-fortified glycopeptide powder and vitamin C, and sonicate, freeze dry and grind to obtain the chelated iron-fortified glycopeptide composition.

2. The method for preparing the chelated iron-containing donkey-hide gelatin glycopeptide composition according to claim 1, characterized in that, The enzymatic hydrolysis conditions in step (1) are: pH 5-6.5, temperature 50-60℃, and hydrolysis time 3-4h; the amount of protease added is 0.1-0.5% of the mass of the gelatin solution; the protease is composed of papain and bromelain in a mass ratio of 1:(1-2); the preparation method of the gelatin solution is: add 2-3 times the amount of water to the gelatin block to melt and boil, keep warm for 15-25min, then add 1-1.5 times the amount of water of the gelatin, and homogenize 2-3 times at 55-70℃ and 125-140MPa.

3. The method for preparing the chelated iron-containing donkey-hide gelatin glycopeptide composition according to claim 1, characterized in that, In step (2), the amount of ethanol solution of ribose derivative added is 0.1-0.5% of the mass of the donkey-hide gelatin peptide hydrolysate; the concentration of ribose derivative in the ethanol solution of ribose derivative is 40-80 mg / g; the conditions for the Maillard reaction are: pH 10-11, temperature 80-90℃, and reaction time 2-3 h; the pH is adjusted to 7-7.

2.

4. The method for preparing the chelated iron-containing donkey-hide gelatin glycopeptide composition according to claim 3, characterized in that, The method for preparing the ribose derivative is as follows: a. Add cypermethrin to anhydrous pyridine, then add methanesulfonyl chloride and stir overnight. Concentrate under vacuum to dryness, then redissolve the residue in N,N-dimethylformamide, add sodium azide, and react at 60-65℃ for 2-6 hours. After the reaction is complete, purify to obtain intermediate 1. The structural formula of intermediate 1 is as follows: b. Add the intermediate 1 and acetylated ribose to a mixture of tetrahydrofuran / water / tert-butanol, add copper sulfate pentahydrate and sodium ascorbate, react overnight at 25-30°C, purify after the reaction is complete to obtain the ribose derivative; The structural formula of the acetylated ribose is as follows: 。 5. The method for preparing the chelated iron-containing donkey-hide gelatin glycopeptide composition according to claim 4, characterized in that, In step a, the ratio of oxaliplatin, methanesulfonyl chloride, and sodium azide is 1 mg: (0.16-0.19) μL: (0.27-0.3) mg; in step b, the molar ratio of intermediate 1, acetylated ribose, copper sulfate pentahydrate, and sodium ascorbate is 1: (1-1.25): (0.2-0.25): (0.4-0.5); and the volume ratio of tetrahydrofuran, water, and tert-butanol in the tetrahydrofuran / water / tert-butanol mixture is 3:1:

1.

6. The method for preparing the chelated iron-containing donkey-hide gelatin glycopeptide composition according to claim 5, characterized in that, The preparation method of the acetylated ribose is as follows: Linoleic acid was added to dichloromethane, followed by 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. After stirring for 30-45 min, (S)-3-amino-5-hexanoate was added and the reaction was allowed to proceed overnight. After the reaction was completed, the mixture was purified to obtain intermediate 2. The structural formula of intermediate 2 is as follows: The intermediate 2,1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added to N,N-dimethylformamide, followed by the addition of 4-dimethylaminopyridine. After stirring and activating for 30-45 min, ribose was added and reacted for 12-24 h. The reaction was terminated by adding water. After purification of the reaction solution, the acetylated ribose was obtained.

7. The method for preparing the chelated iron-containing donkey-hide gelatin glycopeptide composition according to claim 6, characterized in that, step The molar ratio of linoleic acid, (S)-3-amino-5-hexanoate, 4-dimethylaminopyridine, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:(1-1.1):(1.3-1.7):(1.3-1.7); Step The molar ratio of intermediate 2, ribose, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine is 1:(1-1.1):(1.5-2):(1.5-2).

8. The method for preparing the chelated iron-containing donkey-hide gelatin glycopeptide composition according to claim 1, characterized in that, In step (3), the amount of ferrous chloride solution added is 1-5% of the mass of the donkey-hide gelatin peptide solution; the concentration of the ferrous chloride solution is 1-1.5 mol / L; and the stirring reaction time is 30-45 min.

9. The method for preparing the chelated iron-containing donkey-hide gelatin glycopeptide composition according to claim 1, characterized in that, In step (4), the mass ratio of the modified chelated iron-containing glycopeptide powder, β-cyclodextrin, and vitamin C is 1:(18-20):(0.1-0.2); the ultrasonic temperature is 40-60℃ and the time is 20-30min.

10. A chelated iron-containing donkey-hide gelatin glycopeptide composition, characterized in that, It is prepared by the preparation method according to any one of claims 1-9.

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