A nano-collagen fiber iron supplement product and its preparation method

Abstract

This invention proposes a nano-collagen fiber iron supplement product and its preparation method, belonging to the field of food processing technology. The preparation method includes the following steps: (1) dissolving collagen in acetic acid solution and mixing to obtain a collagen solution; (2) dissolving polyphenols in water and adding the obtained polyphenol solution to the collagen solution to obtain a collagen-polyphenol solution; (3) adding laccase to the collagen-polyphenol solution to obtain a polyphenol collagen conjugate; (4) dialyzing the polyphenol collagen conjugate in phosphate solution to obtain a polyphenol collagen composite fiber dispersion; (5) adding iron salt to the polyphenol collagen composite fiber dispersion to obtain the nano-collagen fiber iron supplement product. The preparation method of this invention is simple to operate, and the prepared polyphenol collagen fiber iron complex has a high iron loading and good antioxidant capacity, which helps to reduce oxidative stress, delay the release of iron in the gastrointestinal tract, and thus increase the bioavailability of iron in digestion.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, and in particular relates to a nano-collagen fiber iron supplement product and its preparation method. Background Technology

[0002] Iron is an essential trace element for the human body, playing a vital role in maintaining normal physiological functions. It is also an important cofactor for many enzymes, participating in life processes such as electron transport, DNA, RNA, and protein synthesis, cellular respiration, proliferation and differentiation, and regulation of gene expression. In the nervous system, iron participates in the synthesis of myelin and various neurotransmitters such as norepinephrine, serotonin, and dopamine. Iron is also a functional component of myoglobin and hemoglobin, playing a crucial role in oxygen transport and energy production. Iron deficiency restricts hemoglobin synthesis, leading to the production of hypochromic and microcytic erythrocytes, thus affecting normal development. Furthermore, iron deficiency can cause neurotransmitter synthesis disorders, resulting in delays in behavioral functions such as language and motor balance. Approximately 1.62 billion people worldwide (24.8% of the world's population) suffer from anemia, with iron deficiency being the primary cause. Fortifying food with iron is a sustainable and cost-effective strategy for controlling iron deficiency and iron-deficiency anemia.

[0003] Traditional iron fortification methods involve adding iron fortifiers to food, such as ferrous sulfate, ferrous fumarate, ferric pyrophosphate, and ferric sodium EDTA. However, adding these iron supplements often causes undesirable sensory changes, reducing food acceptability and bioavailability. Furthermore, direct consumption of iron salts may produce side effects such as vomiting or gastric mucosal damage, and may also affect the composition and function of the gut microbiota. Studies have shown that ferrous sulfate can promote the growth of methanogens and lead to a reduction in lactobacilli, an important symbiotic bacterium in infants.

[0004] In recent years, emerging iron delivery systems have attracted widespread attention. Compared with artificially synthesized polymers, food-derived iron delivery carriers are highly regarded for their outstanding characteristics such as biodegradability, biocompatibility, excellent functionality, high nutritional value, low toxicity, and ease of acceptance. For example, amino acids, peptides, proteins, and polysaccharides can chelate or encapsulate iron, thereby improving iron bioavailability and intestinal absorption, reducing sensory problems, minimizing interactions between iron and other components, and preventing direct contact between iron and the gastrointestinal tract, thus reducing side effects. However, complexes formed by amino acids, peptides, and polysaccharides with iron are easily dissociated in the stomach. Therefore, providing a novel iron delivery system that rationally delays iron release is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] This invention addresses the problem of low bioavailability caused by the poor digestibility of iron in the human body by disclosing a nano-collagen fiber iron supplement product and its preparation method.

[0006] This invention proposes a method for preparing a nano-collagen fiber iron supplement product, comprising the following steps:

[0007] (1) Collagen is dissolved in acetic acid solution and mixed to obtain a collagen solution; wherein the collagen has a complete triple helix structure;

[0008] (2) Dissolve polyphenols in water, and add the resulting polyphenol solution to the above collagen solution to obtain a collagen-polyphenol solution; wherein the molecular weight of the polyphenols is less than 500;

[0009] (3) Add laccase to the above collagen-polyphenol solution and mix to obtain polyphenol collagen conjugate;

[0010] (4) Dialyze the above polyphenol collagen conjugate in phosphate solution to obtain a polyphenol collagen composite fiber dispersion;

[0011] (5) Add iron salt to the above polyphenol collagen composite fiber dispersion, mix, and after obtaining polyphenol collagen fiber iron complex, dialyze in deionized water to obtain nano collagen fiber iron supplement product.

[0012] Further, in step (1), the collagen is obtained from animal by-products by acid extraction or enzymatic extraction;

[0013] Preferably, the animal by-products include at least one of cowhide, sheepskin, or pigskin.

[0014] Further, in step (1), the concentration of the acetic acid solution is 0.05 mmol / L;

[0015] In step (1), the concentration of the obtained collagen solution is 0.5-2 mg / mL.

[0016] Furthermore, in step (2), the concentration of the polyphenol solution is 40-240 μmol / L;

[0017] In step (2), the volume ratio of polyphenol solution to collagen solution is 1:1.

[0018] Furthermore, in step (3), the enzyme activity of the laccase is 40 U / mL;

[0019] In step (3), the amount of laccase added is 60 U / g collagen, calculated based on the dry weight of collagen.

[0020] Further, in step (4), the phosphate solution is a 0.1 M Na-phosphate buffer solution containing 100 mM NaCl and pH 6.2.

[0021] Furthermore, in steps (4) and (5), the dialysis is performed using a dialysis bag with a molecular weight cutoff of 8000-14000 Da.

[0022] Further, in step (5), the iron salt includes at least one of ferrous sulfate, ferrous chloride, ferric chloride, ferrous lactate, ferrous gluconate, ferrous fumarate, and ferrous succinate.

[0023] Further, in step (5), iron salt is added to the polyphenol collagen composite fiber dispersion at a mass ratio of collagen to iron ions of 4-6:1.

[0024] This invention also proposes an iron supplement product made of nano-collagen fibers prepared by any of the above-described preparation methods.

[0025] This invention has the following advantages:

[0026] This invention utilizes laccase to catalyze the covalent bonding of polyphenols and collagen, enabling them to self-assemble into fibers in phosphate solution. Iron salts are then mixed with a dispersion of the polyphenol-collagen composite fibers to prepare a polyphenol-collagen fiber-iron complex. The preparation method employed in this invention is simple to operate, and the resulting polyphenol-collagen fiber-iron complex exhibits high iron loading and excellent antioxidant capacity, helping to reduce oxidative stress, delaying iron release in the gastrointestinal tract, and thus increasing the bioavailability of iron during digestion. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 Fluorescence spectra of different concentrations of gallic acid collagen composite fibers provided in Test Example 1 of this invention;

[0029] Figure 2 Fourier transform infrared spectra of gallic acid collagen composite fibers with different concentrations provided in Test Example 1 of this invention;

[0030] Figure 3 This is a diagram showing the proportion of secondary structures of different concentrations of gallic acid collagen composite fibers provided in Test Example 1 of the present invention.

[0031] Figure 4Scanning electron microscope images of gallic acid collagen composite fibers at different concentrations provided in Test Example 1 of this invention (AF represents collagen, collagen fibers, and gallic acid collagen composite fibers with final polyphenol concentrations of 20, 40, 80, and 120 μM, respectively; A is magnified 1000 times; BF is magnified 10000 times).

[0032] Figure 5 The graph shows the iron-binding capacity of different concentrations of gallic acid collagen composite fibers provided in Test Example 1 of this invention (lowercase letters in the graph indicate significant correlation at the p<0.05 level).

[0033] Figure 6 Transmission scanning electron microscope images of the collagen iron complex, collagen fiber iron complex (GA-0-Fib), gallic acid collagen fiber iron complex (GA-20-Fib), and tannin collagen fiber iron complex (T-20-Fib) provided in Test Example 1 of the present invention.

[0034] Figure 7 The graph shows the iron ion release curves during in vitro digestion of different concentrations of gallic acid collagen fiber iron complex provided in Test Example 1 of the present invention. Detailed Implementation

[0035] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the embodiments and features in the embodiments of the present invention can be combined with each other.

[0036] Protein aggregates degrade slowly in the stomach due to their structural characteristics, which helps delay iron release and makes them more suitable as iron delivery carriers. Collagen, as an important component of connective tissue in metazoans, possesses excellent structural properties and various modification properties. Compared to collagen degradation products such as gelatin and collagen peptides, collagen molecules with an intact triple helix structure can self-assemble into fibers under physiological conditions similar to those in vivo. This ordered structure enhances collagen's thermal stability and resistance to enzymatic degradation, making it an effective carrier for binding and delivering iron.

[0037] On one hand, embodiments of the present invention propose a method for preparing a nano-collagen fiber iron supplement product, comprising the following steps:

[0038] (1) Collagen is dissolved in acetic acid solution and mixed to obtain a collagen solution; wherein the collagen has a complete triple helix structure;

[0039] (2) Dissolve polyphenols in water, and add the resulting polyphenol solution to the above collagen solution to obtain a collagen-polyphenol solution; wherein the molecular weight of the polyphenols is less than 500;

[0040] (3) Add laccase to the above collagen-polyphenol solution and mix to obtain polyphenol collagen conjugate;

[0041] (4) Dialyze the above polyphenol collagen conjugate in phosphate solution to obtain a polyphenol collagen composite fiber dispersion;

[0042] (5) Add iron salt to the above polyphenol collagen composite fiber dispersion, mix, and after obtaining polyphenol collagen fiber iron complex, dialyze in deionized water to obtain nano collagen fiber iron supplement product.

[0043] The method for preparing the nano-collagen fiber iron supplement proposed in this invention involves laccase catalysis of the covalent binding of collagen and low-molecular-weight polyphenols, followed by non-covalent assembly into polyphenol-collagen composite fibers in a phosphate solution. In this reaction, the collagen is a soluble, natural collagen with a complete triple helix structure, possessing the characteristic of self-assembling into fibers. Although it can self-assemble into fiber structures, the limited number of its binding sites results in poor iron binding affinity. The addition of low-molecular-weight polyphenols helps collagen self-assemble into fibers of uniform diameter, a fiber morphology that is more conducive to iron ion binding. The resulting nano-collagen fiber iron supplement has a high iron content, antioxidant capacity, helps reduce oxidative stress, delays the release of iron ions in the gastrointestinal tract, and reduces gastrointestinal irritation, showing potential application value in enhancing iron absorption in functional foods.

[0044] In step (1) of this embodiment, collagen with a complete triple helix structure is selected as the reaction raw material. Common forms of collagen in the prior art are mainly of two types: one is collagen fibers peeled off from cowhide, sheepskin, etc., after being fully swollen by soaking in hydrochloric acid, appearing as visible, insoluble fibers with good mechanical properties, generally used as film-forming materials; the other is soluble collagen extracted from animal skin tissue by acid or enzymatic methods, possessing a complete triple helix structure, but generally used for direct iron compounding with poor results. This embodiment of the invention selects soluble collagen with a complete triple helix structure, which can dissolve in acetic acid solution. Utilizing its self-assembly to form fibers, this soluble collagen with a complete triple helix structure is covalently combined with polyphenols under laccase catalysis, and then non-covalently self-assembles to form uniform nanoscale collagen fibers. This fiber state is more conducive to the binding of iron ions.

[0045] In one embodiment of the present invention, in step (1), the collagen is extracted from livestock by-products by acid extraction or enzymatic extraction. The livestock by-products include at least one of cowhide, sheepskin, or pigskin. The acid extraction method [ZHU Q, LI Y, LI S, et al. Fabrication and characterization of acid-soluble collagenstabilized Pickering emulsions [J]. Food Hydrocolloids, 2020, 106, 105875] and the enzymatic extraction method [ZHU S, YUAN Q, YANG M, et al. A quantitative comparable study on multi-hierarchy conformation of acid and pepsin-solubilized collagens from the skin of grass carp (Ctenopharyngodon idella) [J]. Materials Science and Engineering: C, 2019, 96, 446-457] are both conventional methods. This type of collagen has advantages such as wide availability and low cost.

[0046] In one embodiment of the present invention, in step (1), mixing is carried out by stirring.

[0047] In one embodiment of the present invention, in step (1), the concentration of the acetic acid solution is 0.05 mmol / L. The concentration of the resulting collagen solution is 0.5-2 mg / mL. It should be noted that the collagen solution is an acetic acid solution of collagen.

[0048] In one embodiment of the present invention, in step (1), the stirring is specifically carried out under a water bath at 25°C until completely dissolved.

[0049] In step (2) of this embodiment, selecting low molecular weight polyphenols to crosslink with collagen helps collagen self-assemble into fibers of uniform diameter. This fiber morphology is more conducive to the binding of iron ions. When the molecular weight of polyphenols is large, it will cause excessive aggregation of collagen, and iron ions will aggregate in clusters at the intersection of fibers, which is not conducive to the uniform attachment of iron ions.

[0050] In one embodiment of the present invention, in step (2), the polyphenol includes at least one of gallic acid, ferulic acid, caffeic acid, epicatechin, and epigallocatechin gallate. Specifically, the molecular weight of gallic acid is 171, the molecular weight of ferulic acid is 194.18, the molecular weight of caffeic acid is 180.16, the molecular weight of epicatechin is 290.27, and the molecular weight of epigallocatechin gallate is 458.38.

[0051] In one embodiment of the present invention, in step (2), the concentration of the polyphenol solution is 40-240 μmol / L.

[0052] In one embodiment of the present invention, in step (2), the volume ratio of polyphenol solution to collagen solution is 1:1.

[0053] In step (3) of this embodiment, collagen and polyphenols are covalently bound by continuous oscillation under the action of laccase.

[0054] In one embodiment of the present invention, in step (3), the enzyme activity of the laccase is 40 U / mL.

[0055] In one embodiment of the present invention, in step (3), the amount of laccase added is 60 U / g collagen, calculated based on the dry weight of collagen.

[0056] In one embodiment of the present invention, in step (3), mixing is performed by continuous oscillation. Preferably, in step (3), the duration of continuous oscillation is 4 hours. More preferably, in step (3), oscillation is performed using a thermostatic oscillator, wherein the temperature of the thermostatic oscillator is 25 °C and the rotation speed of the thermostatic oscillator is 100 rpm.

[0057] In step (4) of the present invention, the polyphenol collagen conjugate is dialyzed in phosphate solution and self-assembles through non-covalent interaction to form polyphenol collagen composite fibers.

[0058] In one embodiment of the present invention, in step (4), the phosphate solution is a 0.1 M Na-phosphate buffer solution containing 100 mM NaCl and pH 6.2.

[0059] In one embodiment of the present invention, in step (4), the dialysis is performed using a dialysis bag with a molecular weight cutoff of 8000-14000 Da. Preferably, in step (4), the dialysis time is 8-24 h. More preferably, in step (4), the dialysis time is 12 h.

[0060] In step (5) of this embodiment, iron salt is added to the polyphenol collagen composite fiber dispersion. Under continuous shaking, a polyphenol collagen fiber iron complex is formed. Then, it is dialyzed in deionized water to obtain a nano collagen fiber iron supplement product.

[0061] In one embodiment of the present invention, in step (5), the iron salt includes at least one of ferrous sulfate, ferrous chloride, ferric chloride, ferrous lactate, ferrous gluconate, ferrous fumarate, and ferrous succinate.

[0062] In one embodiment of the present invention, in step (5), iron salt is added to the polyphenol collagen composite fiber dispersion at a collagen-to-iron ion mass ratio of 4-6:1. Preferably, in step (5), iron salt is added to the polyphenol collagen composite fiber dispersion at a collagen-to-iron ion mass ratio of 5:1.

[0063] In one embodiment of the present invention, in step (5), the dialysis is performed using a dialysis bag with a molecular weight cutoff of 8000-14000 Da. Preferably, in step (5), the dialysis time is 12-36 h. More preferably, in step (5), the dialysis time is 24 h.

[0064] In one embodiment of the present invention, in step (5), the nano-collagen fiber iron supplement product is stored in liquid form or in powder form after freeze-drying.

[0065] In one embodiment of the present invention, in step (5), mixing is performed by continuous oscillation. Preferably, in step (5), the duration of continuous oscillation is 2-6 hours. Preferably, in step (3), the duration of continuous oscillation is 4 hours. More preferably, in step (5), oscillation is performed using a thermostatic oscillator, wherein the temperature of the thermostatic oscillator is 25°C and the rotation speed of the thermostatic oscillator is 100 rpm.

[0066] On the other hand, this invention also proposes an iron-supplementing product made from nano-collagen fibers prepared by the above-mentioned method. The polyphenol collagen composite fiber prepared by this invention exhibits excellent iron-binding properties. The addition of polyphenols effectively increases the iron ion binding capacity of the collagen fiber. The iron content of the prepared polyphenol collagen fiber iron complex is 5-7.5%, higher than that of heme iron fortifiers. In vitro digestion experiments have demonstrated that the polyphenol collagen composite fiber can effectively delay the release of iron ions in the gastric phase. Simultaneously, this complex possesses good antioxidant capacity, helping to reduce oxidative stress.

[0067] The present invention will now be described in detail with reference to the embodiments and the accompanying drawings.

[0068] Example 1 A method for preparing a nano-collagen fiber iron supplement product specifically includes the following steps:

[0069] (1) Dissolve 100 mg of natural acid-soluble collagen obtained by acid extraction in 100 mL of 0.05 mmol / L acetic acid solution, and stir the solution in a 25 ℃ water bath with a magnetic stirrer until completely dissolved;

[0070] (2) Dissolve gallic acid in deionized water at a concentration of 40 μmol / L, and add 100 mL of the solution to (1) at a volume ratio of 1:1 and stir until homogeneous;

[0071] (3) Using the laccase catalytic oxidation method, according to the dry weight of collagen, add laccase solution (0.15 mL, 40 U / mL) at a concentration of 60 U / g to (2), and shake the solution for 4 h with a constant temperature shaker (25 ℃, 100 rpm) to make it fully mixed;

[0072] (4) Place (3) into an 8000-14000 Da dialysis bag and dialyze for 12 h in 0.1 M Na-phosphate buffer containing 100 mM NaCl and pH 6.2 to form gallic acid-collagen complex fibers;

[0073] (5) Add 100 mg⋅FeSO4⋅7H2O to (4) and stir until completely dissolved. Shake at room temperature for 4 h using a constant temperature shaker (25 ℃, 100 rpm).

[0074] (6) Put (5) into an 8000-14000Da dialysis bag and dialyze in deionized water for 24 h. Dialyze in deionized water to obtain nano-collagen fiber iron supplement product.

[0075] Example 2 A method for preparing a nano-collagen fiber iron supplement product specifically includes the following steps:

[0076] (1) Dissolve 100 mg of natural acid-soluble collagen obtained by acid extraction in 100 mL of 0.05 mmol / L acetic acid solution, and stir the solution in a 25 ℃ water bath with a magnetic stirrer until completely dissolved;

[0077] (2) Dissolve gallic acid in deionized water at a concentration of 80 μmol / L, and add 100 mL of the solution to (1) at a volume ratio of 1:1 and stir until homogeneous;

[0078] (3) Using the laccase catalytic oxidation method, according to the dry weight of collagen, add laccase solution (0.15 mL, 40 U / mL) at a concentration of 60 U / g to (2), and shake the solution for 4 h with a constant temperature shaker (25 ℃, 100 rpm) to make it fully mixed;

[0079] (4) Place (3) into an 8000-14000 Da dialysis bag and dialyze for 12 h in 0.1 M Na-phosphate buffer containing 100 mM NaCl and pH 6.2 to form gallic acid-collagen complex fibers;

[0080] (5) Add 100 mg⋅FeSO4⋅7H2O to (4) and stir until completely dissolved. Shake at room temperature for 4 h using a constant temperature shaker (25 ℃, 100 rpm).

[0081] (6) Put (5) into an 8000-14000Da dialysis bag and dialyze in deionized water for 24 h. Dialyze in deionized water to obtain nano-collagen fiber iron supplement product.

[0082] Example 3 A method for preparing a nano-collagen fiber iron supplement product specifically includes the following steps:

[0083] (1) Dissolve 100 mg of natural acid-soluble collagen obtained by acid extraction in 100 mL of 0.05 M acetic acid solution, and stir the solution in a 25 ℃ water bath with a magnetic stirrer until completely dissolved;

[0084] (2) Dissolve gallic acid in deionized water at a concentration of 160 μmol / L, and add 100 mL of the solution to (1) at a volume ratio of 1:1 and stir until homogeneous;

[0085] (3) Using the laccase catalytic oxidation method, according to the dry weight of collagen, add laccase solution (0.15 mL, 40 U / mL) at a concentration of 60 U / g to (2), and shake the solution for 4 h with a constant temperature shaker (25 ℃, 100 rpm) to make it fully mixed;

[0086] (4) Place (3) into an 8000-14000 Da dialysis bag and dialyze for 12 h in 0.1 M Na-phosphate buffer containing 100 mM NaCl and pH 6.2 to form gallic acid-collagen complex fibers;

[0087] (5) Add 100 mg⋅FeSO4⋅7H2O to (4) and stir until completely dissolved. Shake at room temperature for 4 h using a constant temperature shaker (25 ℃, 100 rpm).

[0088] (6) Put (5) into an 8000-14000Da dialysis bag and dialyze in deionized water for 24 h. Dialyze in deionized water to obtain nano-collagen fiber iron supplement product.

[0089] Example 4 A method for preparing a nano-collagen fiber iron supplement product specifically includes the following steps:

[0090] (1) Dissolve 100 mg of natural acid-soluble collagen obtained by acid extraction in 100 mL of 0.05 mmol / L acetic acid solution, and stir the solution in a 25 ℃ water bath with a magnetic stirrer until completely dissolved;

[0091] (2) Dissolve gallic acid in deionized water at a concentration of 240 μmol / L, and add 100 mL of the solution to (1) at a volume ratio of 1:1 and stir until homogeneous;

[0092] (3) Using the laccase catalytic oxidation method, according to the dry weight of collagen, add laccase solution (0.15 mL, 40 U / mL) at a concentration of 60 U / g to (2), and shake the solution for 4 h with a constant temperature shaker (25 ℃, 100 rpm) to make it fully mixed;

[0093] (4) Place (3) into an 8000-14000 Da dialysis bag and dialyze for 12 h in 0.1 M Na-phosphate buffer containing 100 mM NaCl and pH 6.2 to form gallic acid-collagen complex fibers;

[0094] (5) Add 100 mg⋅FeSO4⋅7H2O to (4) and stir until completely dissolved. Shake at room temperature for 4 h using a constant temperature shaker (25 ℃, 100 rpm).

[0095] (6) Put (5) into an 8000-14000Da dialysis bag and dialyze in deionized water for 24 h. Dialyze in deionized water to obtain nano-collagen fiber iron supplement product.

[0096] Comparative Example 1 A method for preparing a collagen-iron complex (without adding polyphenols, without fiber formation) includes the following steps:

[0097] (1) Dissolve 100 mg of natural acid-soluble collagen obtained by acid extraction in 100 mL of 0.05 mmol / L acetic acid solution, and stir the solution in a 25 ℃ water bath with a magnetic stirrer until completely dissolved;

[0098] (2) Add 100 mL of deionized water to (1) and stir well;

[0099] (3) Add 100 mg⋅FeSO4⋅7H2O to (2) and stir until completely dissolved. Shake at room temperature for 4 h using a constant temperature shaker (25 ℃, 100 rpm).

[0100] (4) Put (3) into an 8000-14000Da dialysis bag and dialyze in deionized water for 24 h to obtain collagen iron complex.

[0101] Comparative Example 2 A method for preparing a collagen fiber iron complex (without adding polyphenols) includes the following steps:

[0102] (1) Dissolve 100 mg of natural acid-soluble collagen obtained by acid extraction in 100 mL of 0.05 mmol / L acetic acid solution, and stir the solution in a 25 ℃ water bath with a magnetic stirrer until completely dissolved;

[0103] (2) Add 100 mL of deionized water to (1) and stir well;

[0104] (3) Place (2) into an 8000-14000 Da dialysis bag and dialyze for 12 h in a 0.1 M Na-phosphate buffer containing 100 mM NaCl and pH 6.2 to form collagen fibers;

[0105] (4) Add 100 mg⋅FeSO4⋅7H2O to (3) and stir until completely dissolved. Shake at room temperature for 4 h using a constant temperature shaker (25 ℃, 100 rpm).

[0106] (5) Put (4) into an 8000-14000Da dialysis bag and dialyze in deionized water for 24 h to obtain collagen fiber iron complex.

[0107] Comparative Example 3 A method for preparing a nano-collagen fiber iron supplement product (with the addition of high molecular weight polyphenols)

[0108] Same as Example 1, except that gallic acid is replaced with tannin, and the molecular weight of tannin is 1701.01.

[0109] Test Example 1

[0110] 1. Intrinsic fluorescence analysis of polyphenol collagen composite fibers

[0111] Gallic acid collagen composite fibers prepared in Examples 1-4 (denoted as GA-20-Fib, GA-40-Fib, GA-80-Fib, and GA-120-Fib, respectively), collagen fibers prepared in Comparative Example 2 (denoted as GA-0-Fib), and 1.0 mg of acid-soluble collagen were dissolved in 2 mL of deionized water. Fluorescent clusters within the protein molecules were scanned using a fluorescence spectrophotometer. The excitation wavelength was 280 nm, the emission wavelength was 290-440 nm, the voltage was fixed at 600 V, the slit width was 5 nm, and the scanning speed was fixed at 240 nm / min.

[0112] Appendix Figure 1The results showed that acid-soluble collagen had a maximum absorption peak at 313 nm and no absorption peak at 350 nm, consistent with the characteristic absorption peaks of collagen. Collagen could initiate self-assembly to form collagen fibers in 0.1 M Na-phosphate buffer containing 100 mM NaCl at pH 6.2. The intrinsic fluorescence intensity decreased after self-assembly, indicating that the exposed amino acids in the molecule participated in the fiber formation process. The addition of gallic acid reduced the fluorescence intensity of collagen fibers, and this decrease gradually increased with increasing gallic acid concentration. This is due to the covalent interaction between the tyrosine residues of collagen and the quinone formed by laccase oxidation of gallic acid. This result indicates that gallic acid is covalently linked to collagen via laccase-catalyzed oxidation.

[0113] 2. Fourier Transform Infrared Spectroscopy (FTIR) Analysis of Polyphenol Collagen Composite Fibers

[0114] The FTIR spectra of gallic acid collagen composite fibers were obtained using a Thermo Nicolet Avatar 370 FTIR spectrometer (Vector 22, Bruker, Germany) equipped with a DTGS KBr detector (Thermo, Shanghai, China). First, 1 mg of gallic acid collagen composite fibers prepared in Examples 1-4 (denoted as GA-20-Fib, GA-40-Fib, GA-80-Fib, and GA-120-Fib, respectively), collagen fibers prepared in Comparative Example 2 (denoted as GA-0-Fib), and 1 mg of acid-soluble collagen were added to a mortar and pestle, followed by 150 mg of potassium bromide (KBr). The mixture was then ground finely and transferred to a mold for tableting. Each sample was subjected to FTIR spectra from 4000 cm⁻¹. -1 Up to 500 cm -1 The scans were performed and analyzed using OMNIC 8.2 software. The relative content of secondary structures was obtained by baseline correction, smoothing, deconvolution, and second derivative fitting of the amide bands, and by calculating the area of ​​each subpeak.

[0115] Appendix Figure 2 The results showed that the amide A band of acid-soluble collagen was at 3424.36 cm⁻¹. -1 The absorption peak at 2927.67 cm⁻¹ indicates that the NH group participates in hydrogen bonding, thereby maintaining the stability of the triple helix structure. -1 The amide B band observed at [location] represents asymmetric stretching of CH2. Compared to collagen, [protein] self-assembled at 3404.89 cm⁻¹. -1The presence of an absorption peak in the amide A band indicates that hydrogen bonds are formed during collagen assembly. The addition of gallic acid did not cause a significant wavenumber shift in the infrared spectrum of collagen fibers. By performing Fourier reversible convolution and Gaussian fitting on the amide I band, the proportions of β-sheets, L-polyproline helices, and β-turns of collagen, collagen fibers, and collagen composite fibers with different concentrations of gallic acid were obtained. (Appendix) Figure 3 The results showed that the proportions of β-sheets, L-proline helices, and β-turns in collagen were 42%, 26%, and 32%, respectively. After self-assembly, the proportion of L-proline helices decreased to 23%, while the proportion of β-turns increased. This indicates that collagen assembly is driven by hydrophobic interactions. At gallic acid concentrations of 20-40 μmol / L, the proportion of β-turns in the composite fibers increased. With increasing gallic acid concentration, the fibers transitioned to β-sheets. This suggests that the addition of gallic acid has a rearrangement effect on the secondary structure of the assembled collagen fibers.

[0116] 3. Observation of the morphology of polyphenol collagen composite fibers using scanning electron microscopy.

[0117] Trace amounts of gallic acid collagen composite fibers (denoted as GA-20-Fib, GA-40-Fib, GA-80-Fib, and GA-120-Fib, respectively) prepared in Examples 1-4, collagen fibers (denoted as GA-0-Fib) prepared in Comparative Example 2, and acid-soluble collagen were respectively attached to the sample stage with conductive adhesive. Then, all samples were sputter-coated with gold for 3 min under argon atmosphere and scanned using SEM (SU3800, Hitachi, Japan) at an acceleration voltage of 7.0 kV.

[0118] Appendix Figure 4 The results showed that the freeze-dried collagen molecules exhibited a lamellar membrane structure with interconnected filaments. The fibrils formed after collagen assembly at 25 ℃ and pH 6.2 exhibited uneven thickness, with coarse fibrils formed by the aggregation of fine fibrils. With increasing gallic acid content, the diameter of the gallic acid-collagen composite fibers increased and became more uniform. This indicates that within this addition range, gallic acid promoted collagen self-assembly, forming nano-collagen fibers with a diameter of 70-90 nm.

[0119] 4. Analysis of the iron-binding capacity of polyphenol collagen composite fibers

[0120] The iron-loaded gallic acid collagen composite fibers prepared in Examples 1-4 (denoted as GA-20-Fib, GA-40-Fib, GA-80-Fib, and GA-120-Fib, respectively), the collagen fiber iron complex prepared in Comparative Example 2 (denoted as GA-0-Fib), and the collagen iron complex in Comparative Example 1 were heated at 90 °C for 30 min, and the iron-binding capacity of the samples was determined using phenanthroline. 0.5 mL of the test solution was taken, and 0.1 mL of 10% ascorbic acid solution was added, and the reaction was allowed to proceed for 1 h. Then, 0.4 mL of 10% ammonium acetate solution was added, followed by 0.1 mL of 12 mM phenanthroline solution. The mixture was allowed to stand at room temperature for 10 min, and the absorbance at 562 nm was read using a microplate reader. Water was used as a blank control. Iron(II) standard solutions (0-50 μM) were used as a standard curve.

[0121] Appendix Figure 5 The results showed that the iron-binding capacity of the collagen-iron complex in Comparative Example 1 was 1.6%, while the iron-binding capacity of the collagen fiber-iron complex increased to 5.0% after self-assembly. This indicates that collagen fibers have a stronger affinity for iron ions than collagen molecules. The addition of gallic acid increased the iron-binding capacity of collagen fibers. When the gallic acid concentration was above 40 μmol / L, the iron-binding capacity was significantly enhanced. When the polyphenol concentration in the system was 120 μmol / L, the iron-binding capacity reached 8.1%, which is three to four times that of heme iron (1.0-2.5%). This indicates that iron ions not only bind to collagen fibers but also to gallic acid. The greater the amount of gallic acid added, the more iron ions bind to it, and the ortho-dihydroxy structure of gallic acid can form a coordination bond with iron ions.

[0122] 5. Transmission electron microscopy analysis of polyphenol collagen fiber iron complex

[0123] The dispersions of the gallic acid-collagen fiber iron complex (GA-20-Fib) of Example 1 (with a polyphenol concentration of 20 μmol / L), the collagen-iron complex of Comparative Example 1, and the collagen-iron complex (GA-0-Fib) of Comparative Example 2 were subjected to transmission electron microscopy (TEM). 10 μL of the diluted sample was deposited on a carbon-supported membrane on a copper grid for 1 min, then rinsed with ultrapure water and dried. The iron distribution of the complexes was analyzed by TEM at 100 kV and EDS.

[0124] Appendix Figure 6The results show that, from left to right, the samples are: Comparative Example 1 (collagen-iron complex), Comparative Example 2 (collagen-fiber-iron complex), Example 1 (gallic acid-collagen-fiber-iron complex), and Comparative Example 3 (tannin-collagen-fiber-iron complex). From top to bottom, the images show the structural diagrams of the corresponding samples, the distribution of iron in the samples as shown by dark-field scanning energy dispersive spectroscopy (EDS), and the combination diagram of the two. The collagen-iron complex exhibits a fine, irregular rope-like structure, and iron ions can be seen attached to collagen molecules under high-angle annular dark-field scanning. The collagen-fiber-iron complex exhibits a fibrous structure of varying thickness, with obvious iron ion aggregation on the larger diameter collagen fibers. The gallic acid-collagen-fiber-iron complex exhibits a regular, ordered long fibrous structure, with iron ions relatively uniformly attached to the fibers. The tannin-collagen-fiber-iron complex exhibits a fibrous aggregation state. This is because tannin is a highly polymerized compound among polyphenols with a large molecular weight, which promotes excessive aggregation of collagen. Iron ions aggregate in clusters at the fiber intersections, indicating that the addition of tannin is not conducive to the formation of a regular, ordered fibrous structure in collagen and is not conducive to the uniform attachment of iron ions. In summary, the addition of gallic acid is beneficial for collagen to form an effective carrier capable of loading iron ions.

[0125] 6. Detection of iron ion release during in vitro digestion

[0126] The release of iron ions during simulated in vitro gastrointestinal digestion was analyzed using the dialysis diffusion method for the gallic acid collagen fiber iron complexes prepared in Examples 1-4 (denoted as GA-20-Fib, GA-40-Fib, GA-80-Fib, and GA-120-Fib, respectively), the collagen fiber iron complex prepared in Comparative Example 2 (denoted as GA-0-Fib), and the collagen iron complex in Comparative Example 1. Prepare the gastric and intestinal phase electrolytes according to Table 1. Add 2 mL of sample dispersion (5 mg / mL) to the dialysis bag (MWCO 8000-14000Da) and completely immerse it in 50 mL of simulated gastric fluid (pH 2.0, 3.2 mg / mL pepsin dissolved in the gastric phase electrolyte) for 2 h. Then transfer the dialysis bag to 50 mL of simulated intestinal fluid (pH 7.0, 10 mg / mL trypsin, 2.5 mg / mL bile salts dissolved in the intestinal phase electrolyte) for 3 h. Dialyze at 37 °C with continuous shaking at 150 rpm. Take the dialysate every 30 min and detect the iron ion content in the dialysate at 562 nm using the phenanthridine colorimetric method. Use Equation (1) to fit the zero-order model of the kinetic release curve of iron ions.

[0127] Q t = Q0+k0t (1)

[0128] In the formula Q t t represents the amount of iron ions released within time t; Q0 represents the initial amount of the sample in the solution; and k0 represents the zero-order release constant.

[0129] Table 1. Preparation of Electrolyte for In Vitro Digestion

[0130]

[0131] Appendix Figure 7 The results showed that the collagen iron complex released 82% of its iron ions after 30 min of gastric digestion, and reached 93% after 2 h of digestion. The collagen fiber iron complex released 74% of its iron ions after 30 min of gastric digestion, and further released 84.5% with increasing digestion time. Compared to the former two, the gallic acid collagen fiber iron complex released iron ions at a relatively slow rate. The addition of polyphenols effectively reduced the iron ion release rate in the gastric phase, and this rate decreased with increasing polyphenol concentration. After 2 h of gastric digestion, the iron ion release rates of 20-120 μmol / L gallic acid collagen fiber iron complexes were 58.2%, 40.3%, 36.9%, and 21.0%, respectively. This is because gallic acid collagen fibers, as protein aggregates, degrade slowly in the stomach, which delays iron release, thus retaining more iron ions for the intestines. Table 2 shows the iron ion release kinetics of different samples fitted using a zero-order model, and the apparent release constant k0 was calculated using a linear regression equation. During the rapid release phase, the release constant k0 of the gallic acid collagen fiber iron complex was significantly lower than that of the collagen iron complex and the collagen fiber iron complex, indicating that pepsin exhibits a delayed effect on the hydrolysis of the polyphenol collagen fiber iron complex.

[0132] In simulated intestinal fluid, the iron ion release rates of the collagen iron complex, collagen fiber iron complex, and gallic acid collagen fiber iron complex were all relatively stable. The release constant k0 of the gallic acid collagen fiber iron complex increased, mainly due to further hydrolysis of the loose protein structure by trypsin in the simulated intestinal fluid. The total iron ion release from the collagen iron complex during simulated gastrointestinal digestion was 96.35%, the total iron ion release from the collagen fiber iron complex was 92%, and the iron ion release rates of the 20-120 μmol / L gallic acid collagen fiber iron complex were 68.98%, 50.25%, 48.37%, and 25.45%, respectively. Therefore, compared with the collagen iron complex and collagen fiber iron complex, the tightly assembled gallic acid collagen fiber iron complex is more conducive to delayed dissociation in an acidic environment, and its retention in the intestine is more beneficial for the slow release of iron ions.

[0133] Table 2. R values ​​of the zero-order model kinetic curves for iron ion release. 2 and release constant k0

[0134]

[0135] 7. Analysis of the antioxidant capacity of polyphenol collagen fiber iron complex

[0136] DPPH radical scavenging ability: 2,2-diphenyl-1-picrylhydrazine (DPPH, 1 mg) was dissolved in 20 mL of 95% ethanol to obtain the DPPH working solution. 2 mL of the DPPH working solution was mixed into 1 mL of the sample solution and reacted in the dark for 30 minutes. The absorbance of the solution at 519 nm was measured. The DPPH radical scavenging activity was calculated using equation (1):

[0137] DPPH free radical scavenging rate (%) = (A0-A1) / A0×100 Equation (2)

[0138] Where A0 is the absorbance of DPPH without the sample at 519 nm, and A1 is the absorbance of DPPH with the sample at 519 nm.

[0139] ABTS radical scavenging ability: 10 mg of 2,2'-nitro-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was dissolved in 2.6 mL of potassium persulfate solution to prepare ABTS stock solution, and the reaction was carried out at room temperature in the dark for 16 h. The solution was diluted with deionized water to an absorbance of 0.70 at 734 nm. Another 1 mL of sample solution was diluted to a certain concentration, and 3 mL of ABTS solution was added. The mixture was shaken for 30 s and allowed to react at room temperature in the dark for 60 min, and the absorbance at 734 nm was measured. The ABTS radical scavenging activity was calculated using formula (3):

[0140] ABST free radical scavenging rate (%) = (A0-A1) / A0×100 Equation (3)

[0141] Where A0 and A1 represent the absorbance of ABTS at 734 nm with and without the sample, respectively.

[0142] Table 3 shows the antioxidant capacity of the gallic acid-collagen fiber iron complex. The DPPH and ABTS free radical scavenging results indicate that the collagen iron complex, collagen fiber iron complex, and gallic acid-collagen fiber iron complex all possess antioxidant capabilities. With increasing gallic acid concentration, the antioxidant capacity of the gallic acid-collagen fiber iron complex gradually increases, suggesting that the prepared nano-collagen fiber iron supplement helps reduce oxidative stress.

[0143] Table 3. Antioxidant capacity of collagen iron complex, collagen fiber iron complex, and gallic acid collagen fiber iron complex.

[0144]

[0145] Note: Different lowercase letters represent significant differences (P < 0.05).

[0146] Based on the comprehensive analysis of the results in 1-7 above, the iron supplement product prepared by this invention was characterized. Endogenous fluorescence experiments showed a covalent interaction between polyphenols and collagen; infrared spectroscopy showed that the addition of polyphenols altered the composition of the secondary structure of collagen fibers; scanning electron microscopy showed that the prepared polyphenol-collagen composite fibers had increased and more uniform diameters, ranging from 70-90 nm; iron-binding capacity experiments showed that the iron-binding amount of the composite fibers increased (5-8%) with increasing polyphenol concentration; transmission electron microscopy showed that iron ions could be uniformly attached to the polyphenol-collagen composite fibers; in vitro digestion experiments showed that the addition of polyphenols effectively delayed the release of iron ions in the gastric phase; and antioxidant capacity experiments showed that the polyphenol-collagen fiber iron complex had good antioxidant capacity. Table 4 shows the content of each component in the iron supplement product prepared by this invention: collagen content was 89.3-94.5%, polyphenol content was 0.5-3.2%, and iron content was 5.0-7.5%. In summary, the polyphenolic collagen fibers prepared by this invention can serve as an effective carrier for iron delivery, and the polyphenolic collagen fiber iron complex can effectively improve the bioavailability of iron in the human digestive process as a functional food.

[0147] Table 4. Content of various components in nano-collagen fiber iron supplement products

[0148]

[0149] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a nano-collagen fiber iron supplement product, characterized in that, Includes the following steps: (1) Collagen is dissolved in acetic acid solution and mixed to obtain a collagen solution; wherein the collagen has a complete triple helix structure; (2) Dissolve polyphenols in water and add the resulting polyphenol solution to the above collagen solution to obtain a collagen-polyphenol solution; the polyphenol is gallic acid; the concentration of the polyphenol solution is 40-240 μmol / L; the volume ratio of the polyphenol solution to the collagen solution is 1:

1. (3) Add laccase to the above collagen-polyphenol solution and mix to obtain a polyphenol collagen conjugate; the enzyme activity of the laccase is 40 U / mL; the amount of laccase added is 60 U / g collagen, calculated based on the dry weight of collagen. (4) Dialyze the above polyphenol collagen conjugate in phosphate solution to obtain a polyphenol collagen composite fiber dispersion; (5) Add iron salt to the above polyphenol collagen composite fiber dispersion, mix, and after obtaining polyphenol collagen fiber iron complex, dialyze in deionized water to obtain nano collagen fiber iron supplement product; wherein, the iron salt includes ferrous sulfate; the iron salt is added to the polyphenol collagen composite fiber dispersion at a collagen to iron ion mass ratio of 4-6:

1.

2. The preparation method according to claim 1, characterized in that, In step (1), the collagen is obtained from animal by-products by acid extraction or enzymatic extraction.

3. The preparation method according to claim 1, characterized in that, In step (1), the concentration of the acetic acid solution is 0.05 mmol / L; In step (1), the concentration of the obtained collagen solution is 0.5-2 mg / mL.

4. The preparation method according to claim 1, characterized in that, In step (4), the phosphate solution is a 0.1 M Na-phosphate buffer solution containing 100 mM NaCl and pH 6.

2.

5. The preparation method according to claim 1, characterized in that, In steps (4) and (5), the dialysis is performed using a dialysis bag with a molecular weight cutoff of 8000-14000 Da.

6. The nano-collagen fiber iron supplement product prepared by the preparation method according to any one of claims 1-5.

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