Protein fiber-oxidized polysaccharide compound, iron-loaded compound and preparation methods of protein fiber-oxidized polysaccharide compound and iron-loaded compound

Abstract

The invention discloses a protein fiber-oxidized polysaccharide compound, an iron-loaded compound and preparation methods of the protein fiber-oxidized polysaccharide compound and the iron-loaded compound. The preparation method comprises the following steps: hydrolyzing whey protein isolate with alkaline protease to obtain whey protein isolate enzymolysis powder; mixing the whey protein isolate and the enzymolysis powder according to a mass ratio of 1: 1 to prepare a hydrolyzed protein fiber solution; sodium periodate oxidizes dextran to prepare an oxidized dextran solution; and grafting the oxidized dextran solution and the hydrolyzed protein fiber solution under the action of sodium cyanoborohydride to prepare the protein fiber-oxidized polysaccharide compound. Loading ferric chloride on the protein fiber-oxidized polysaccharide compound, and reducing by NaBH4 to obtain an iron-loaded compound; the invention provides a novel method for controllably preparing the protein fiber-oxidized polysaccharide compound at normal temperature, and the iron loading capacity is as high as 1: 1; the iron-loaded compound nano iron is more stable in valence state, higher in oxidation resistance and good in biological safety and bioavailability, the cell absorption effect is 1.33 times that of a conventional iron supplementing agent ferrous sulfate, and the iron supplementing effect is safe and efficient.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and particularly relates to a preparation method and use of a protein fiber-oxidized polysaccharide complex and an iron-loaded complex. Background Art

[0002] Iron deficiency is the most common micronutrient deficiency in the world. There are about 1.6 billion iron-deficient people, accounting for more than 30% of the world's population. The design and development of iron supplements are crucial. The first-generation iron supplements are inorganic ferrous salts, and the second-generation iron supplements are small-molecule organic acid iron salts. Both generations of iron supplements have obvious irritation to the human gastrointestinal tract. Free ferrous ions are easily oxidized and endogenous free radicals are generated, causing cell membrane damage, and are gradually fading out of sight. Ferrous ion is a common iron supplement component, but ferrous iron is easily oxidized to ferric iron, and the oxidized ferric iron is not easily absorbed by the human body. At the same time, the generated hydroxyl free radicals will cause peroxidation and apoptosis. Various iron supplements on the current market do not pay enough attention to effectively stabilizing ferrous iron to improve its absorption rate and reducing the potency loss caused by oxidation. Although ferrous iron is an ideal supplementary form due to its higher bioavailability, its easy oxidation characteristic requires special attention to stability issues in the formulation design to ensure the maximization of the iron supplement effect. Research shows that improving the antioxidant performance of the iron transport system and stabilizing ferric ions in the divalent state that is easily absorbed by the human body helps maintain the stability of iron and promote iron absorption. Therefore, there is an urgent need to develop a macromolecular composite carrier with high antioxidant properties, which can maintain the valence stability of ferrous ions while improving the bioavailability of iron through the macromolecular structure of the carrier and promoting the absorption of iron ions by the human body. Macromolecular complex iron supplement systems such as polysaccharide iron, protein iron, and fiber iron have the advantages of good stability, high safety, strong antioxidant ability, high bioavailability, etc., and are gradually being taken seriously in the research of iron loading. At the same time, these macromolecular composite iron supplements can reduce human gastrointestinal irritation and improve the subsequent iron absorption ability in the human body, and have the potential to become a new generation of iron supplements.

[0003] Whey protein isolate is a common by-product in the production of cheese. It is widely used in food processing due to its rich nutritional value and important biological functions. In particular, whey protein isolate amyloid fibrils have quite excellent antioxidant activity and emulsifying properties. However, under low pH conditions, the active side chains of the amino groups of whey protein isolate tend to be protonated more, with low affinity for cations, which leads to a weakened chelation property with metal ions, thus greatly reducing the iron-binding ability of whey protein. Therefore, in practical applications, it is necessary to strengthen its binding ability with metal ions by modifying and decorating whey protein isolate.

[0004] Enzymatic hydrolysis of proteins is a common method for protein modification. Existing studies have shown that precise enzymatic hydrolysis can enhance the fiber-forming ability, effectively shorten the formation time of mature fibers, and endow the formed fibers with high antioxidant activity and strong metal ion chelating activity. Meanwhile, by grafting and modifying the enzymatically treated protein fibers with polysaccharides and utilizing the unique structure of protein fiber-oxidized polysaccharides, the chelating ability of protein fibers with metal ions can be greatly improved, enabling the iron-loaded complex to exhibit excellent antioxidant and biological activities simultaneously.

[0005] Therefore, developing a new process method for controllable preparation of protein fiber-oxidized polysaccharide complexes and deeply studying its core technology in iron delivery, using protein fiber-oxidized polysaccharides to encapsulate and deliver iron, so that nano-iron maintains higher bioavailability in the composite system and reduces irritation to the intestine, can lay a foundation for the development and application of new healthy iron supplements. Summary of the Invention

[0006] Aiming at the technical problems of existing iron supplements such as poor taste, strong irritation, poor stability, low iron carrying capacity, and poor iron supplementation effect, the present invention provides a protein fiber-oxidized polysaccharide complex, an iron-loaded complex, and a preparation method thereof. The present invention prepares the complex through a crosslinking reaction, constructs a new protein fiber-polysaccharide complex with high antioxidant properties, and enables whey protein isolate and dextran to have high grafting degree, high antioxidant properties, and strong stability under simple reaction conditions. Meanwhile, the iron-loaded complex prepared with the protein fiber-oxidized polysaccharide complex provided by the present invention as a carrier has a high iron loading capacity, stable iron ion valence state, long absorption time in the intestine, little irritation and harm to the human intestine, and high bioavailability.

[0007] To achieve the above object, the present invention is realized through the following technical solutions.

[0008] A preparation method of a protein fiber-oxidized polysaccharide complex, the method comprising the following steps: (1) Hydrolyze whey protein isolate with alkaline protease to obtain whey protein isolate enzymatic hydrolysis powder; mix whey protein isolate with whey protein isolate enzymatic hydrolysis powder at a mass ratio of 1:1, hydrate under acidic conditions, heat and stir to react, and cool to obtain a hydrolyzed protein fiber solution; (2) Oxidize dextran under the action of sodium periodate to obtain an oxidized dextran solution; (3) Graft the oxidized dextran solution and the hydrolyzed protein fiber solution under the action of sodium cyanoborohydride at 0-4°C to obtain a protein fiber-oxidized polysaccharide graft product.

[0009] Further, preferably, step (1) is carried out according to the following steps: Dissolve whey protein isolate in deionized water to prepare a whey protein isolate solution with a concentration of 3 - 4% (weight / volume). Adjust the pH to 8, heat to 95 - 100 °C and hold for 10 - 15 min. Then add alkaline protease and heat at 50 - 55 °C for 2 - 4 h. Centrifuge to obtain the supernatant and lyophilize to get the hydrolyzed whey protein isolate powder. Mix the hydrolyzed whey protein isolate powder and whey protein isolate powder at a mass ratio of 1:1 to prepare a dispersion with a concentration of 20 - 25 mg / mL. Adjust the pH to 1.5 - 2 with acid and place at 0 - 4 °C for 8 - 15 h for hydration. Then heat and stir at 85 - 90 °C for 4 - 8 h and cool to obtain the hydrolyzed protein fiber solution (WPH); Further, preferably, step (2) is carried out as follows: Dissolve dextran and sodium periodate in acetate buffer so that the concentration of dextran is 2 - 3 mg / mL and the concentration of sodium periodate is 10 - 15 mM; stir the reaction in the dark for 1 - 2 h, then add ethylene glycol to terminate the reaction, and dialyze to remove small molecules to obtain the oxidized dextran solution; Further, preferably, step (3) is carried out as follows: Mix the oxidized dextran solution, hydrolyzed protein fiber solution and sodium cyanoborohydride in a ratio of solute mass of 1:0.8 - 1.2:0.8 - 1.2, stir evenly, and react at 0 - 4 °C for 5 - 8 h to obtain the protein fiber - oxidized polysaccharide graft product; In step (1), the mass dosage of alkaline protease is preferably 4 - 5% of the mass of whey protein isolate.

[0010] In step (1), mix the hydrolyzed whey protein isolate powder and whey protein isolate powder at a mass ratio of 1:1, adjust the pH to 1.5 - 2 with acid, and place at 0 - 4 °C for 8 - 15 h for hydration. Hydration can improve the hydration properties of proteins, promote the unfolding of protein structures, increase the solubility of proteins, and enhance the functionality of proteins, providing a good basis for subsequent fiber production.

[0011] In step (2), the dextran is preferably dextran with a molecular weight between 40 - 50 kDa.

[0012] In step (2), the concentration of acetate buffer is preferably 20 - 30 mM.

[0013] In step (2), the dosage of ethylene glycol is 3 - 4 μL of ethylene glycol added per 1 mg of sodium periodate.

[0014] In step (2), the dialysis is generally carried out twice with 0.15 M sodium chloride solution for 1-2 hours each time, then dialyzed with phosphate buffer solution at pH = 7.4 for 3-4 hours, and finally dialyzed with water for 8-15 hours.

[0015] In step (3), sodium cyanoborohydride can promote the formation of stable bonds between aldehyde-containing molecules and amine-containing molecules, thereby realizing the coupling of proteins and other molecules.

[0016] In step (3), the solute mass ratio of oxidized dextran solution, hydrolyzed protein fiber solution and sodium borohydride is preferably 1:1:1.

[0017] In step (3), the reaction time is preferably 6 hours.

[0018] The present invention also provides a protein fiber-oxidized polysaccharide complex prepared by the above method.

[0019] The described protein fiber-oxidized polysaccharide complex can be used to carry active substances that are easily oxidized.

[0020] The protein fiber-oxidized polysaccharide complex of the present invention can be used to prepare antioxidant functional foods, health foods or drugs.

[0021] Furthermore, the protein fiber-oxidized polysaccharide complex is used to prepare a protein fiber-oxidized polysaccharide iron-loaded complex, and the method of this application is: loading ferric chloride on the protein fiber-oxidized polysaccharide complex and reducing it with NaBH4 to obtain the protein fiber-oxidized polysaccharide iron-loaded complex.

[0022] The present invention also provides a preparation method of a protein fiber-oxidized polysaccharide iron-loaded complex, and the method includes the following steps: (1) Hydrolyze whey protein isolate with alkaline protease to obtain whey protein isolate hydrolysate powder; mix whey protein isolate and whey protein isolate hydrolysate powder in a mass ratio of 1:1, hydrate under acidic conditions, heat and stir for reaction, and cool to obtain a hydrolyzed protein fiber solution; (2) Carry out an oxidation reaction on dextran under the action of sodium periodate to obtain an oxidized dextran solution; (3) Graft the oxidized dextran solution and the hydrolyzed protein fiber solution under the action of sodium cyanoborohydride at 0-4 °C to obtain a protein fiber-oxidized polysaccharide complex; (4) Load ferric chloride on the protein fiber-oxidized polysaccharide complex and reduce it with NaBH4 to obtain a protein fiber-oxidized polysaccharide iron-loaded complex.

[0023] Preferably, step (4) is carried out according to the following steps: Mix the protein fiber-oxidized polysaccharide graft product with a ferric chloride hexahydrate solution at a solute mass ratio of 1:0.8 - 1.2, stir at room temperature for 20 - 50 min, then slowly add NaBH4, let it stand for 1 - 2 h, and purify by dialysis with water to obtain the protein fiber-oxidized polysaccharide iron-loaded complex.

[0024] In the step (4), the solute mass ratio of the protein fiber-oxidized polysaccharide graft product to the ferric chloride hexahydrate solution refers to the mass ratio of the solid mass in the protein fiber-oxidized polysaccharide graft product to the mass of the ferric chloride hexahydrate solute in the ferric chloride hexahydrate solution.

[0025] Furthermore, preferably in the step (4), the solute mass ratio of the protein fiber-oxidized polysaccharide graft product to the ferric chloride hexahydrate solution is preferably 1:1.

[0026] In the step (4), the mass ratio of ferric chloride hexahydrate to NaBH4 is 5 - 6:1, preferably 5:1.

[0027] The present invention also provides the protein fiber-oxidized polysaccharide iron-loaded complex prepared by the above method. The protein fiber-oxidized polysaccharide iron-loaded complex is a protein fiber-dextran nanoiron complex and can be used as a nanoiron iron supplement.

[0028] The protein fiber-oxidized polysaccharide iron-loaded complex of the present invention can be used for preparing iron-supplementing drugs.

[0029] The protein fiber-oxidized polysaccharide iron-loaded complex of the present invention can be used for preparing anti-anemia drugs.

[0030] The present invention provides a protein fiber-oxidized polysaccharide composite (WPH-DES) and a protein fiber-oxidized polysaccharide iron-loaded complex carrying iron ions (abbreviated as WPH-DEX-Fe). The protein fiber in the protein fiber-oxidized polysaccharide complex is formed by proportionally mixing hydrolyzed whey protein isolate and unhydrolyzed whey protein isolate, followed by fibrillation treatment to form a protein fiber solution. The protein fiber solution has a large number of free amino groups, and Schiff base reactions can occur between the amino groups and carbonyl groups. To enable more sites on the polysaccharide to covalently graft with the hydrolyzed protein fiber, the present invention uses sodium periodate to oxidize glucose to produce the corresponding compound. Subsequently, the oxidized low-molecular-weight dextran (dextran 40) is used for precise graft modification of the protein fiber, and the protein fiber and the oxidized polysaccharide are stably grafted through Schiff base reactions to form a protein fiber-oxidized polysaccharide complex, thereby further enhancing the iron chelating ability by the addition of the polysaccharide. The protein fiber-oxidized polysaccharide iron-loaded complex reduces ferric chloride to divalent iron ions under the action of sodium borohydride and binds them to the protein fiber-oxidized polysaccharide complex, successfully constructing a protein fiber-oxidized polysaccharide-iron ternary complex. The iron-loaded complex developed in the present invention changes the tertiary structure of the protein fiber, improves the antioxidant property and stability of the protein fiber system, can be directly absorbed by the digestive tract without being converted into other forms, greatly improves the iron utilization rate, and can also continue to promote the transport and distribution of iron in the body after being absorbed.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The present invention uses natural whey protein isolate and polysaccharide as raw materials, has good biocompatibility and low cytotoxicity, and is suitable for long-term human consumption.

[0032] (2) The present invention uses enzymatically hydrolyzed whey protein isolate and oxidized polysaccharide as raw materials, and through a series of preparation steps such as enzymatic hydrolysis, oxidation, grafting, and reduction, covering various chemical modifications, constructs a composite system with high antioxidant property and high iron loading capacity. The protein fiber with high antioxidant property can effectively stabilize the valence state of divalent iron and reduce the oxidative loss of iron ions during storage and transportation.

[0033] (3) The present invention uses the free amino groups of the enzymatically hydrolyzed protein fiber to crosslink with the carbonyl groups released by oxidized dextran, and the formed complex has a grafting degree as high as 86% under the condition of 4°C, significantly improving the structural stability of the system and enhancing the anti-degradation ability of the system under different environmental conditions.

[0034] (4) The protein fiber-oxidized polysaccharide complex constructed in the present invention has a ferric ion loading ratio of the grafted product as high as 1:1, which is significantly higher than that of traditional iron supplements, and can provide a higher iron content at a lower dose, improving the iron supplementation effect. At the same time, the nanometerization treatment of the composite system ensures the iron transport capacity and stability, meeting the market demand for high-concentration iron supplementation. The protein fiber-oxidized polysaccharide complex provided by the present invention can not only meet the scenarios with high requirements for iron supplementation effect and safety, but also be used as an emulsifier, antioxidant, and carrier for anti-anemia drugs (such as the development of functional foods, health foods, and pharmaceuticals), with broader functional expandability and application prospects.

[0035] (5) The iron-loaded complex provided by the present invention has good biosafety, low cytotoxicity, little intestinal irritation, and high bioavailability. The cell absorption effect is significantly improved compared with conventional ferrous salts. Taking the cell absorption of ordinary ferrous sulfate as the standard, the cell absorption of the protein fiber-oxidized polysaccharide iron-loaded complex is 1.33 times that of ferrous sulfate. Description of the Drawings

[0036] Figure 1 It is a characterization diagram of the controllable preparation and binding mechanism of the protein fiber and oxidized polysaccharide complex at different reaction times: Figure A is the grafting degree diagram at different reaction times, Figure B is the comparison diagram of the contents of the primary and final products of the protein fiber-oxidized polysaccharide grafted product, Figure C is the ultraviolet full-spectrum scanning spectrum of the protein fiber-oxidized polysaccharide grafted product at different reaction times, and Figure D is the infrared spectrum of the protein fiber-oxidized polysaccharide grafted product at different reaction times.

[0037] Figure 2 It is a detection diagram of the antioxidant capacity of the protein fiber-oxidized polysaccharide complex: Figure A is the comparison diagram of the reducing power of whey protein fiber after being treated by different methods, and Figure B is the comparison diagram of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability of whey protein fiber after being treated by different methods.

[0038] Figure 3 It is an X-ray photoelectron spectroscopy (XPS) spectrum of the divalent iron content in the iron-loaded complex of whey protein fiber after different treatment methods.

[0039] Figure 4 It is a transmission electron microscope microscopic display diagram of the iron-loaded protein fiber-oxidized polysaccharide complex under different pH and heating conditions.

[0040] Figure 5 It is a promotion effect diagram of the iron-loaded complex of protein fiber on the growth of Caco-2 cells and the ferritin content diagram during the cell absorption process after different treatment methods. Among them, Figure A is the relative cell survival rate (%) at different concentrations, and Figure B is the ferritin content (ng / mg) diagram during the cell absorption process.

[0041] Figure 6 Iron ion release concentration diagrams of protein fiber-loaded iron complexes after different treatments in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Specific embodiments

[0042] To better understand the present invention, the present invention will be further described below in conjunction with embodiments. There are many successful embodiments of the present invention, and six specific embodiments are listed below, but the scope of protection required by the present invention is not limited to these embodiments only.

[0043] Example 1: Controllable preparation of protein fiber-oxidized polysaccharide complex and characterization of binding mechanism Sample preparation: (1) Adjust the whey protein isolate dispersion (4% weight / volume ratio) to pH = 8 with alkali, heat it in a water bath at 100 °C for 10 min, then add alkaline protease (the enzyme mass dosage is 4% of the protein mass), heat it at 50 °C for 150 min, and then centrifuge. Take the supernatant and freeze-dry it into whey protein isolate hydrolysate powder. Mix the whey protein isolate hydrolysate powder and whey protein isolate powder in a mass ratio of 1:1, dissolve it in water to prepare a dispersion with a concentration of 20 mg / ml, adjust the pH to 2 with acid, and hydrate it overnight at 4 °C. Then heat it in a water bath at 90 °C for 5 h, quickly transfer it to an ice-water bath to cool, and cool it to room temperature to terminate the self-assembly of the microstructure, obtaining a hydrolyzed protein fiber solution, denoted as WPH.

[0044] (2) Dissolve dextran (dextran 40) and sodium periodate in 20 mM acetate buffer so that the concentration of dextran is 2.5 mg / ml and the concentration of sodium periodate is 10 mM. Stir it on a magnetic stirrer for 90 min, react in the dark, and then add ethylene glycol to terminate the reaction (add 3.73 μl of ethylene glycol for every 1 mg of sodium periodate). Then dialyze it successively with 0.15 M sodium chloride solution (1 h each time, dialysis twice) and pH = 7.4 phosphate buffer (3 h each time, dialysis once), and then dialyze it with water overnight to obtain an oxidized dextran solution.

[0045] (3) Mix the oxidized dextran solution, hydrolyzed protein fiber solution and sodium cyanoborohydride (NaBH3CN) in a solute mass ratio of 1:1:1, stir evenly, and react at 4 °C for 2, 3, 4, 5, 6, 7, 12 h respectively to obtain protein fiber-oxidized polysaccharide graft products at different reaction times, denoted as WPH-DEX.

[0046] (4) Mix the protein fiber-oxidized polysaccharide graft product with FeCl3∙6H2O at a mass ratio of solute (the mass of ferric chloride hexahydrate is regarded as the solute) of 1:1, stir at room temperature for 0.5 h, then slowly add NaBH4, and the mass ratio of sodium borohydride to ferric chloride hexahydrate is 1:5. Let it stand for 1 h. After stabilization, dialyze overnight with pure water to obtain a protein fiber-oxidized polysaccharide composite iron-loaded system, denoted as WPH-DEX-Fe.

[0047] Control group: Whey protein isolate was dissolved in water to prepare a dispersion with a concentration of 20 mg / ml, adjusted to pH = 2 with acid, and hydrated overnight at 4 °C. Then heat it in a water bath at 90 °C for 5 h, quickly transfer it to an ice-water bath to cool, and cool to room temperature to terminate the self-assembly of the microstructure to obtain a whey protein isolate fiber solution, denoted as WPI.

[0048] Operate according to the above step (3) to prepare a whey protein isolate fiber-oxidized polysaccharide graft product, denoted as WPI-DEX.

[0049] The grafting reaction situation diagram of protein fiber and oxidized polysaccharide at different reaction times is as Figure 1 shown. Among them, Figure A is the grafting degree diagram at different reaction times. Add 4 mL of OPA reagent to 200 μL of the sample and mix well, and incubate at 35 °C for 2 min. Use 200 μL of ultrapure water instead of the sample as a control, and measure the absorbance at 340 nm. Draw a standard curve with lysine as the standard and calculate the free amino group content. The calculation formula of the grafting degree (DG) is as follows: , and the highest grafting degree was measured to be 86% at 6 h. It shows that the synergistic modification method of the present invention not only enhances the structural stability of the composite system but also improves its anti-degradation ability under different environmental conditions.

[0050] Figure B is a comparison diagram of the primary and ultimate product contents of the protein fiber-oxidized polysaccharide graft product. Dilute the sample with 0.1% sodium dodecyl sulfate to a protein concentration of 2 mg / mL, and measure the absorbance at 294 nm and 420 nm using a UV spectrophotometer, respectively marked as A 294 and A 420。The primary products include unreacted proteins and polysaccharides, as well as the initially formed proteoglycan graft copolymers. The ultimate product is the brownish-black melanoidin substance. The test results show that the reaction has been controlled at the primary stage. The high content of primary products indicates that the synergistic modification method of the present invention has controlled the Maillard reaction at the primary stage. The early grafting can reduce the interference with the active sites of proteins, thereby enhancing the stability and emulsifying properties of the proteoglycan system. In addition, the test results show that the protein fiber-oxidized polysaccharide has a high grafting degree at 4 °C, and the grafting degree is the highest when the reaction is carried out for 6 h. The high grafting degree can improve the stability of the system. Therefore, the preferred reaction time of the present invention is 6 h. All subsequent experiments use the protein fiber-oxidized polysaccharide graft product prepared by reacting for 6 h as the raw material, denoted as WPH-DEX.

[0051] Figure C shows the full-band scanning spectrogram of the ultraviolet spectrum of the protein fiber-oxidized polysaccharide graft product at different reaction times. As shown in Figure C, WPI shows two main absorption peaks in the wavelength range of 220-500 nm. The first absorption peak is located at 220-240 nm and is caused by the characteristic ultraviolet absorption of peptide bonds. As the grafting reaction proceeds, the intensity of this peak weakens and redshifts, presumably due to the reaction between the carbonyl group at the reducing end of the polysaccharide and the protein to form structures such as Schiff bases. The second absorption peak increases in intensity in the range of 260-320 nm, but the maximum absorption wavelength remains unchanged. It is speculated that the grafting of dextran causes the exposure of aromatic amino acid residues in WPH, enhancing the ultraviolet absorption. The redshift and intensity change of the absorption peak indicate that the tertiary structure of the protein has changed, indicating that the polysaccharide (dextran) in the composite system prepared by the present invention has been successfully grafted onto the protein fiber.

[0052] Figure D shows the infrared spectrogram of the protein fiber-oxidized polysaccharide graft product at different reaction times. The absorption intensity of the amide I band (C=O stretching vibration, 1645 cm -1 ) in the whey protein isolate-dextran graft product is slightly lower than that of pure WPI fibers, which may be attributed to the reduction in carbonyl content and the gradual polymerization into larger molecules. However, the Schiff base of the reaction product has an absorption peak at this position, resulting in some absorption at a wavenumber of 1654 cm -1 . At the same time, the absorption peak of the amide II band (N-H bending vibration, 1529 cm -1 ) weakens significantly, indicating that a large amount of carbonyl and amino groups are lost between dextran and WPI fibers, and a carbonyl-amino reaction has occurred. The amide I band (1645 cm -1 ) and amide III band (1240 cm -1)There were significant changes after dextran grafting, indicating that the tertiary structure of the protein had been altered by the polysaccharide. The composite system prepared in the present invention confirmed the efficient grafting of dextran to protein fibers. This high grafting degree not only enhanced the structural stability of the composite system but also improved its anti-degradation ability under different environmental conditions.

[0053] Example 2: Detection of the antioxidant capacity of protein fiber-oxidized polysaccharide complexes Comparison charts of the reducing power and free radical scavenging ability of whey protein isolate fibers WPI, WPH, and WPH-DEX treated by different methods are as Figure 2 shown, where Figure A is the reducing power and Figure B is the DPPH free radical scavenging ability.

[0054] For the determination of reducing power, 50 μL of protein dispersion (WPI, WPH, or WPH-DEX) was pipetted into an enzyme-linked immunosorbent assay (ELISA) plate, and FeCl3·6H2O (0.1 M) was added so that the mass ratio of Fe to protein was 1:5. Subsequently, an excess of 1,10-phenanthroline was added, and the absorbance at 512 nm was measured after 15 min, which was designated as OD 512 nm. For the determination of DPPH scavenging ability, a 0.1 mmol / l DPPH methanol solution was prepared and stored in the dark. The protein fiber solutions treated by different methods were mixed with the DPPH methanol solution and left in the dark for 1 h. The absorbance of the mixture at 517 nm was immediately measured and designated as A1. At the same time, an equal volume of distilled water was mixed with the DPPH-methanol solution, and the absorbance A2 was measured as a blank control. An equal volume of the sample solution was mixed with the methanol solution, and the absorbance A3 was measured as a control. The following formula was used to calculate the free radical scavenging rate: When analyzing the reducing power and DPPH free radical scavenging ability of protein fibers after different treatments, it was observed that the reducing power and scavenging ability of the protein fiber samples treated by enzymatic hydrolysis and polysaccharide grafting were significantly better than those of pure protein fibers. This indicates that the composite prepared in the present invention has high antioxidant properties and helps to stabilize divalent iron ions.

[0055] Example 3: Detection of the divalent iron content in the protein fiber-oxidized polysaccharide iron-loaded complex The whey protein isolate fiber solution was mixed with FeCl3∙6H2O at a mass ratio of the solute (the mass of ferric chloride hexahydrate was recorded as the solute) of 1:1, stirred at room temperature for 0.5 h, and then NaBH4 was slowly added at a mass ratio of sodium borohydride to iron of 1:5. It was allowed to stand for 1 h, and after stabilization, it was dialyzed against pure water overnight to obtain the whey protein isolate fiber iron-loaded complex, designated as WPI-Fe.

[0056] Replace the whey protein fiber solution with the hydrolyzed protein fiber solution prepared in step (1) of Example 1 to obtain a hydrolyzed whey protein fiber iron complex, denoted as WPH-Fe.

[0057] X-ray photoelectron spectroscopy (XPS) analysis was performed on newly prepared whey protein isolate fiber-iron (WPI-Fe), hydrolyzed whey protein fiber-iron (WPH-Fe), and hydrolyzed whey protein fiber-dextran-iron (WPH-DEX-Fe) nanopowder samples to study their composition. The results are as Figure 3 shown. XPS identifies the composition of iron and distinguishes the iron core and outer layer of the nanoparticles. In this experiment, the relative contents of iron(II) and iron(III) in the samples were analyzed to demonstrate that under the conditions of co-modification, the antioxidant capacity, the ability to stabilize divalent iron, and the iron loading capacity for divalent iron were enhanced. Through XPS analysis, the peak area ratios of iron(II) to iron(III) in the three samples were determined to be 23:77, 49:51, and 77:23, respectively. This indicates that the protein fiber-oxidized polysaccharide iron complex after co-modification treatment has higher antioxidant properties and can stabilize the valence state of iron, thus promoting the absorption and utilization of iron in the gastrointestinal tract.

[0058] Example 4: Characterization of the iron transport stability of the protein fiber-oxidized polysaccharide complex As Figure 4 shown in the morphological observation results of the protein fiber-oxidized dextran iron complex under a transmission electron microscope (TEM). It can be clearly observed from the figure that as the pH value increases from 3 to 9, the dispersion state of the protein fibers changes significantly, showing a trend from dispersion to aggregation.

[0059] Under the condition of a lower pH value (such as pH = 3), the protein fiber-oxidized dextran iron complex presents a relatively clear nanofiber structure, with a relatively uniform distribution among the fibers and no obvious aggregation phenomenon. This may be because under acidic conditions, the charge state on the surface of the protein fibers helps to maintain the electrostatic repulsion between the fibers, thus maintaining the dispersion stability of the fibers.

[0060] As the pH value gradually increases to neutral or alkaline conditions (such as pH = 7 and 9), it can be observed that obvious aggregation of the protein fibers begins to occur. This aggregation may be due to the reduction of the charge on the surface of the protein fibers under neutral conditions, resulting in a weakened electrostatic repulsion and an enhanced attraction between the fibers, thus promoting the aggregation of the fibers.

[0061] When the pH value further increases to 9, the aggregation phenomenon of protein fibers is more serious, forming larger aggregates. Under alkaline conditions, the structure of protein fibers may change. For example, the β-sheet structure may transform into other forms, further promoting the aggregation of fibers. In addition, metal ions may have stronger interactions with protein fibers and polysaccharides at higher pH values, which may also promote the aggregation of fibers.

[0062] After heating the protein fiber-oxidized dextran iron complex (product pH = 4 - 5) at 90 °C for two hours, the sample still maintains the fiber morphology without obvious changes, indicating that the sample shows good thermal stability, providing stable conditions for subsequent processing.

[0063] It shows that even under extreme pH conditions and high-temperature heating, the protein fibers after the co-modification strategy still maintain the fiber morphology, indicating that the protein fiber iron complex has higher stability.

[0064] Example 5: Determination of the bioavailability of protein fiber-oxidized polysaccharide iron complex The promotion effect diagram of the protein fiber-loaded iron complex on the growth of Caco-2 cells and the ferritin content diagram during cell absorption after different treatments are as Figure 5 shown, where Figure 5 -A is the relative cell survival rate (%) at different concentrations, Figure 5 -B is the ferritin content (ng / mg) diagram during cell absorption. The non-toxicity of the iron-loaded system is verified through Caco-2 cell toxicity and cell absorption, and it has a significant effect on cell iron absorption.

[0065] For cytotoxicity assessment, a standard CCK-8 experiment was used to measure the cytotoxicity of Caco-2 cells. The steps of the CCK-8 experiment are as follows: 5.0 × 10 3 cells were seeded in each well of a 96-well plate and cultured at 37 °C and 5% CO2 for 24 h. The cell culture medium was removed, and the samples digested and diluted with the culture medium were added to each well. Then the cells were cultured for another 24 h in an environment containing 5% CO2. 10 μl of CCK-8 solution was added to each well. After incubating in the incubator for an appropriate time, the absorbance at 450 nm was measured using a microplate reader to determine the cell survival rate.

[0066] Cell absorption assessment. Once a complete monolayer of Caco-2 cells is formed, it can be used to evaluate the bioavailability of iron. First, discard the original culture medium in the culture plate, and then add 100 μl of freshly prepared sample solution to each well of the six-well plate. Subsequently, add 600 μl of DMEM medium, and set up three replicate wells for each group. Then incubate the cells in a cell culture incubator for 24 h. After discarding the medium, wash the upper chamber twice with pre-cooled PBS. Add an appropriate amount of RIPA lysis buffer on ice to collect cell lysates. Measure the protein content according to the instructions of the total protein (BCA) detection kit and the ferritin (Fe) detection kit. The bioavailability of iron is determined by dividing the concentration of ferritin by the concentration of total protein.

[0067] Figure 5 -A results show that all samples prepared by the present invention have a positive effect on the growth and division of Caco-2 cells at this concentration. However, as the concentration increases, only the protein fiber grafted with dextran has a cell growth exceeding 100% at 0.5 mg / ml, proving that at increased concentrations, the protein fiber-oxidized polysaccharide iron complex provided by the present invention has no toxic effect on cell growth.

[0068] Figure 5 -B results show that the protein fiber-oxidized polysaccharide iron complex has a significant promoting effect on cell growth. After being absorbed by Caco-2 cells, the ferritin content of the protein fiber grafted with polysaccharide is much higher than that of the non-grafted protein fiber, and it is 1.33 times that of the conventional iron supplement ferrous sulfate, proving that the protein fiber-oxidized polysaccharide iron complex of the present invention has better bioavailability.

[0069] Example 6: Evaluation of the digestion ability of the protein fiber-oxidized polysaccharide iron complex in the in vitro gastrointestinal tract The iron ion release concentration diagrams of the protein fiber iron complexes WPI-Fe, WPH-Fe, and WPH-DEX-Fe treated by different methods in simulated gastric juice (SGF) and intestinal juice (SIF) are as Figure 6 shown. Simulated gastric juice (SGF): Weigh 0.2 g of NaCl, dissolve it in 100 ml of water, adjust the pH to 2.0 with 1 mol / l HCl, and add an appropriate amount of pepsin (enzyme concentration is 10 mg / ml).

[0070] Simulated gastric digestion: Prepare a protein fiber-loaded iron complex solution with a concentration of 2.5 mg / ml. Take 20 ml of this solution and place it in a conical flask, and adjust the pH to 2.0 with 1 mol / l HCl. Then heat the flask to 37 °C and maintain for 10 min. Subsequently, add 20 ml of simulated gastric fluid (SGF), shake well, seal it, and oscillate on a shaker at 37 °C for reactions of 30 min, 60 min, 90 min, and 120 min respectively. After the reaction is completed, measure the Fe 2+ content.

[0071] Simulated intestinal fluid (SIF): Weigh 0.68 g of KH2PO4 and 0.062 g of NaOH, dissolve them in 100 ml of water, and then adjust the pH to 6.8 with 1 mol / l NaOH. Add an appropriate amount of trypsin with an enzyme concentration of 10 mg / ml. For the simulated intestinal digestion process, after the simulated gastric digestion is completed, adjust the pH to 6.8 with 1 mol / l NaOH and preheat at 37 °C for 5 min. Then add 4 ml of simulated intestinal fluid (SIF), shake well, seal it, and oscillate on a shaker at 37 °C for reactions of 30 min, 60 min, 90 min, and 120 min respectively. After the reaction is completed, quickly heat the reaction mixture to 90 °C to inactivate the enzyme and maintain for 10 min. After cooling to room temperature, measure the Fe 2+ content.

[0072] Figure 6 The results show that the content of divalent iron in the WPH-DEX complex increases in simulated gastric fluid (pH = 2.0). Because pepsin hydrolyzes the protein fiber, destroying the stability of the peptide-iron chelate, the fiber is temporarily dispersed; at the same time, the strong acidic environment leads to a weakening of the binding ability between iron ions and the fiber, causing the iron ions to dissociate from the fiber, resulting in the released divalent iron ions being temporarily in a free state. In contrast, due to the poor stability of the composite system of WPI and WPH, the content of divalent iron is low and it cannot be stably released. After entering the simulated intestinal fluid, the solubility of iron ions decreases in a neutral or alkaline environment, and it is easier to recombine with the fiber. Therefore, divalent iron ions recombine with the fiber to form a stable peptide-iron complex in a neutral environment, reducing free divalent iron ions, improving bioavailability and reducing gastrointestinal irritation. At the same time, because the fiber has good antioxidant properties, it protects divalent iron from oxidation, which is beneficial to intestinal absorption. It shows that the novel composite system prepared by the present invention can enable the protein fiber and iron ions to recombine in the intestine to form an iron-loaded complex, and these iron-loaded complexes can then be absorbed by the intestine, improving bioavailability, and at the same time reducing the damage and irritation that free divalent iron ions may cause to the human gastrointestinal tract.

[0073] The above embodiments are intended to illustrate the embodiments disclosed in the present invention and should not be construed as limiting the present invention. In addition, various modifications listed herein and changes in the methods and compositions of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in connection with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, all such modifications that are obvious to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. A method for preparing a protein fiber-oxidized polysaccharide complex, characterized in that The method comprises the following steps: (1) Hydrolyze whey protein isolate with alkaline protease to obtain hydrolyzed whey protein isolate powder; mix whey protein isolate with the hydrolyzed whey protein isolate powder at a mass ratio of 1:1, hydrate under acidic conditions, heat and stir for reaction, and cool to obtain a hydrolyzed protein fiber solution; (2) Conduct an oxidation reaction on dextran under the action of sodium periodate to obtain an oxidized dextran solution; (3) Graft the oxidized dextran solution and the hydrolyzed protein fiber solution under the action of sodium cyanoborohydride at 0 - 4 °C to obtain a protein fiber-oxidized polysaccharide complex.

2. The preparation method of the protein fiber-oxidized polysaccharide complex according to claim 1, characterized in that The reaction steps of step (1) are as follows: Dissolve whey protein isolate in deionized water to obtain a whey protein isolate solution with a weight-volume concentration of 3 - 4%, adjust the pH to 8, heat to 95 - 100 °C and keep warm for 10 - 15 min, then add alkaline protease, heat at 50 - 55 °C for 2 - 4 h, centrifuge to take the supernatant, freeze-dry to obtain hydrolyzed whey protein isolate powder, mix whey protein isolate with the hydrolyzed whey protein isolate powder at a mass ratio of 1:1 to obtain a dispersion with a concentration of 20 - 25 mg / mL, add acid to adjust the pH value to 1.5 - 2, place at 0 - 4 °C for 8 - 15 h for hydration, and then heat and stir for reaction at 85 - 90 °C for 4 - 8 h, and cool to obtain a hydrolyzed protein fiber solution; The reaction steps of step (2) are as follows: Dissolve dextran and sodium periodate in acetate buffer solution so that the concentration of dextran is 2 - 3 mg / mL and the concentration of sodium periodate is 10 - 15 mM; stir in the dark for 1 - 2 h, then add ethylene glycol to terminate the reaction, and dialyze to remove small molecules to obtain an oxidized dextran solution; The reaction steps of step (3) are as follows: Mix the oxidized dextran solution, the hydrolyzed protein fiber solution and sodium cyanoborohydride in a ratio of solute mass ratio of 1:0.8 - 1.2:0.8 - 1.2, stir evenly, and react at 0 - 4 °C for 5 - 8 h to obtain a protein fiber-oxidized polysaccharide complex.

3. A protein fiber-oxidized polysaccharide complex prepared by the method according to claim 1 or 2.

4. Use of the protein fiber-oxidized polysaccharide complex according to claim 3 as a carrier for transporting easily oxidizable active substances.

5. Use of the protein fiber-oxidized polysaccharide complex according to claim 3 in the preparation of antioxidant functional foods, health foods or drugs.

6. A method for preparing a protein fiber-oxidized polysaccharide iron-loaded complex, characterized in that The method comprises the following steps: (1) Hydrolyze whey protein isolate with alkaline protease to obtain hydrolyzed whey protein isolate powder; mix whey protein isolate with the hydrolyzed whey protein isolate powder at a mass ratio of 1:1, hydrate under acidic conditions, heat and stir for reaction, and cool to obtain a hydrolyzed protein fiber solution; (2) Conduct an oxidation reaction on dextran under the action of sodium periodate to obtain an oxidized dextran solution; (3) Graft the oxidized dextran solution and the hydrolyzed protein fiber solution under the action of sodium cyanoborohydride at 0 - 4 °C to obtain a protein fiber-oxidized polysaccharide complex; (4) Load the protein fiber-oxidized polysaccharide complex with ferric chloride and reduce it with NaBH4 to obtain a protein fiber-oxidized polysaccharide iron-loaded complex.

7. The method according to claim 6, wherein The reaction step of step (4) is as follows: Mix the protein fiber-oxidized polysaccharide graft product with a ferric chloride hexahydrate solution at a solute mass ratio of 1: 0.8-1.2, stir at room temperature for 20-50 min, then slowly add NaBH4, let stand for 1-2 h, and purify by dialysis with water to obtain a protein fiber-oxidized polysaccharide iron-loaded complex.

8. A protein fiber-oxidized polysaccharide iron-loaded complex prepared by the method according to claim 6 or 7.

9. Use of the protein fiber-oxidized polysaccharide iron-loaded complex according to claim 8 as an iron supplement.

10. Use of the protein fiber-oxidized polysaccharide iron-loaded complex according to claim 8 in the preparation of an iron supplement drug or an anti-anemia drug.

Images