A protein fiber-oxidized polysaccharide complex, a ferric-loaded complex, and a preparation method thereof

Abstract

The application discloses a protein fiber-oxidized polysaccharide compound, an iron-loaded compound and a preparation method thereof. The preparation method is as follows: whey protein isolate is hydrolyzed by alkaline protease to obtain whey protein isolate enzymolysis powder; the whey protein isolate and the enzymolysis powder are mixed at a mass ratio of 1:1 to prepare a hydrolyzed protein fiber solution; oxidized dextran solution is prepared by oxidizing dextran with sodium periodate; the oxidized dextran solution and the hydrolyzed protein fiber solution are grafted under the action of sodium cyanoborohydride to obtain the protein fiber-oxidized polysaccharide compound. The protein fiber-oxidized polysaccharide compound is loaded with ferric chloride and reduced by NaBH4 to obtain the iron-loaded compound. The application provides a novel method for preparing the protein fiber-oxidized polysaccharide compound at room temperature, and the iron loading amount is as high as 1:1; the iron-loaded compound has more stable nano-iron valence, higher oxidation resistance, good biological safety and biological availability, and the cell absorption effect is 1.33 times that of a conventional iron supplement ferrous sulfate, and the iron supplement effect is safe and efficient.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomaterials, and particularly relates to a protein fiber-oxidized polysaccharide complex, a preparation method of an iron-loaded complex and uses thereof. BACKGROUND

[0002] Iron deficiency is the most common micronutrient deficiency in the world, with about 1.6 billion people suffering from iron deficiency, accounting for more than 30% of the world's population. The design and development of iron supplements are crucial. The first generation of iron supplements is inorganic ferrous salt, and the second generation is small-molecule organic acid iron salt. Both generations of iron supplements have obvious irritation to the gastrointestinal tract, and free ferrous ions are easily oxidized and produce endogenous free radicals, causing cell membrane damage, and are gradually fading from the field of view. Ferrous iron 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, and the hydroxyl radicals produced simultaneously can cause peroxidation and apoptosis. The various iron supplements on the current market do not pay enough attention to the stability of ferrous iron to improve its absorption rate and reduce the loss of efficacy due to oxidation. Although ferrous iron is an ideal supplement due to its higher bioavailability, its easy oxidation requires special attention to stability in the formulation design to ensure the maximization of iron supplement effect. Studies have shown that improving the antioxidant properties of iron transport systems and stabilizing iron ions in the ferrous state that is easily absorbed by the human body can help maintain iron stability and promote iron absorption. Therefore, it is urgent to develop a high-antioxidant macromolecular complex carrier that can maintain the stability of ferrous ions while improving the bioavailability of iron through the macromolecular structure of the carrier to promote the absorption of iron ions by the human body. Macromolecular complex iron supplement systems such as polysaccharide iron, protein iron, and fiber iron have good stability, high safety, strong antioxidant capacity, and high bioavailability, and are gradually gaining attention in iron carrier research. At the same time, these macromolecular complex iron supplements can reduce gastrointestinal irritation and improve subsequent iron absorption 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 and is widely used in food processing due to its rich nutritional value and important biological functions, especially whey protein isolate amyloid fibers, which have excellent antioxidant activity and emulsifying properties. However, under low pH conditions, the active side chains of whey protein isolate amino groups tend to be protonated, with low affinity for cations, which reduces their chelation properties with metal ions, thereby significantly reducing the ability of whey protein to bind iron. Therefore, in practical applications, it is necessary to modify and modify whey protein isolate to enhance its binding capacity with metal ions.

[0004] Enzymatic hydrolysis of protein is a common method for protein modification. Existing studies have shown that precise enzymatic hydrolysis can enhance the fiber formation ability, effectively shorten the formation time of mature fibers, and make the fibers show high antioxidant activity and strong metal ion chelating activity after fiber formation. At the same time, the protein fiber and polysaccharide subjected to enzymatic hydrolysis are grafted and modified, and the unique structure of protein fiber-oxidized polysaccharide can greatly improve the chelating capacity of protein fiber and metal ions, so that the iron-loaded complex exhibits excellent antioxidant activity and biological activity.

[0005] Therefore, a new process method for preparing protein fiber-oxidized polysaccharide complex is developed, and the core technology of iron delivery is further studied. The use of protein fiber-oxidized polysaccharide for encapsulating and delivering iron enables nano-iron to maintain a higher bioavailability in the complex system and reduce the irritation to the intestinal tract, which lays a foundation for the development and application of new healthy iron supplements. SUMMARY

[0006] In view of the technical problems of the existing iron supplements, such as poor taste, strong irritation, poor stability, low iron carrying capacity, and poor iron supplement effect, the present application provides a protein fiber-oxidized polysaccharide complex, an iron-loaded complex and a preparation method thereof. The present application prepares the complex through cross-linking reaction, constructs a new protein fiber-polysaccharide complex with high antioxidant activity, and enables whey protein isolate and dextran to have high grafting degree, high antioxidant activity and strong stability under simple reaction conditions. At the same time, the iron-loaded complex prepared by using the protein fiber-oxidized polysaccharide complex as a carrier has high iron carrying capacity, stable iron ion valence, long absorption time in the intestinal tract, small irritation and harm to the human intestinal tract, and high bioavailability.

[0007] In order to achieve the above-mentioned purpose, the present application is realized by the following technical scheme.

[0008] A preparation method of a protein fiber-oxidized polysaccharide complex, the method comprising the following steps:

[0009] (1) hydrolyzing whey protein isolate with alkaline protease to obtain whey protein isolate hydrolysis powder; mixing whey protein isolate and whey protein isolate hydrolysis powder according to a mass ratio of 1:1, hydrating under acidic conditions, and heating and stirring to react to prepare a hydrolyzed protein fiber solution;

[0010] (2) oxidizing dextran under the action of sodium periodate to prepare an oxidized dextran solution;

[0011] (3) grafting the oxidized dextran solution and the hydrolyzed protein fiber solution under the action of sodium cyanoborohydride at 0-4℃ to prepare a protein fiber-oxidized polysaccharide grafting product.

[0012] Further, the step (1) is preferably performed according to the following steps:

[0013] The whey protein isolate is dissolved in deionized water to prepare a whey protein isolate solution with a concentration of 3-4% (weight / volume), the pH is adjusted to 8, and the solution is heated to 95-100°C for 10-15 min. Then, alkaline protease is added, and the solution is heated at 50-55°C for 2-4 h. The supernatant is obtained by centrifugation, and the whey protein isolate hydrolyzed powder is obtained by freeze-drying. The whey protein isolate hydrolyzed powder is mixed with the whey protein isolate powder at a mass ratio of 1:1 to prepare a dispersion solution with a concentration of 20-25 mg / mL. The pH is adjusted to 1.5-2 by adding acid, and the solution is hydrated at 0-4°C for 8-15 h. Then, the solution is heated and stirred at a temperature of 85-90°C for 4-8 h to prepare a hydrolyzed protein fiber solution (WPH).

[0014] Further, preferably, the step (2) is performed according to the following steps:

[0015] The dextran and sodium periodate are dissolved in an acetic acid buffer to obtain a dextran concentration of 2-3 mg / mL and a sodium periodate concentration of 10-15 mM. The solution is stirred in the dark for 1-2 h, and then the reaction is terminated by adding ethylene glycol. The small molecules are removed by dialysis to obtain an oxidized dextran solution.

[0016] Further, preferably, the step (3) is performed according to the following steps:

[0017] The oxidized dextran solution, the hydrolyzed protein fiber solution, and sodium cyanoborohydride are mixed at a solute mass ratio of 1:0.8-1.2:0.8-1.2, uniformly stirred, and reacted at 0-4°C for 5-8 h to obtain a protein fiber-oxidized polysaccharide grafted product.

[0018] In the step (1), the mass of the alkaline protease is preferably 4-5% of the mass of the whey protein isolate.

[0019] In the step (1), the whey protein isolate hydrolyzed powder is mixed with the whey protein isolate powder at a mass ratio of 1:1, the pH is adjusted to 1.5-2 by adding acid, and the solution is hydrated at 0-4°C for 8-15 h. The hydration can improve the hydration properties of the protein, promote the unfolding of the protein structure, improve the solubility of the protein, and enhance the functionality of the protein, thereby providing a good foundation for subsequent fiber production.

[0020] In the step (2), the dextran is preferably dextran with a molecular weight of 40-50 kDa.

[0021] In the step (2), the concentration of the acetic acid buffer is preferably 20-30 mM.

[0022] In the step (2), the amount of ethylene glycol added is 3-4 µl per 1 mg of sodium periodate.

[0023] In the step (2), the dialysis is generally performed twice with 0.15 M sodium chloride solution for 1-2 h, then with pH = 7.4 phosphate buffer for 3-4 h, and finally with water for 8-15 h.

[0024] In the 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.

[0025] In the step (3), the solute mass ratio of the oxidized dextran solution, the hydrolyzed protein fiber solution and sodium borohydride is preferably 1:1:1.

[0026] In the step (3), the reaction time is preferably 6 h.

[0027] The present application also provides a protein fiber-oxidized polysaccharide complex prepared by the above method.

[0028] The protein fiber-oxidized polysaccharide complex can be used to carry active substances that are easily oxidized.

[0029] The protein fiber-oxidized polysaccharide complex of the present application can be used to prepare antioxidant functional foods, health foods or medicines.

[0030] Further, the protein fiber-oxidized polysaccharide complex is used to prepare a protein fiber-oxidized polysaccharide iron-loaded complex, and the application method is as follows: the protein fiber-oxidized polysaccharide complex is loaded with ferric chloride, and reduced by NaBH4 to obtain the protein fiber-oxidized polysaccharide iron-loaded complex.

[0031] The present application also provides a preparation method of a protein fiber-oxidized polysaccharide iron-loaded complex, which comprises the following steps:

[0032] (1) whey protein isolate is hydrolyzed by alkaline protease to obtain whey protein isolate hydrolysis powder; whey protein isolate and whey protein isolate hydrolysis powder are mixed in a mass ratio of 1:1, hydrated under acidic conditions, heated and stirred to react, and cooled to obtain a hydrolyzed protein fiber solution;

[0033] (2) dextran is subjected to oxidation reaction under the action of sodium periodate to obtain an oxidized dextran solution;

[0034] (3) the oxidized dextran solution and the hydrolyzed protein fiber solution are grafted under the action of sodium cyanoborohydride at 0-4℃ to obtain a protein fiber-oxidized polysaccharide complex;

[0035] (4) the protein fiber-oxidized polysaccharide complex is loaded with ferric chloride, and reduced by NaBH4 to obtain a protein fiber-oxidized polysaccharide iron-loaded complex.

[0036] Preferably, the step (4) is performed as follows:

[0037] The protein fiber-oxidized polysaccharide graft product is mixed with the ferric chloride solution at a solute mass ratio of 1:0.8-1.2, stirred at room temperature for 20-50 min, then slowly added with NaBH4, and left to stand for 1-2 h, and then purified by water dialysis to obtain the protein fiber-oxidized polysaccharide iron-loaded complex.

[0038] In the step (4), the solute mass ratio of the protein fiber-oxidized polysaccharide graft product to the ferric chloride solution refers to the mass ratio of the solid in the protein fiber-oxidized polysaccharide graft product to the ferric chloride solute in the ferric chloride solution.

[0039] Further, preferably in the step (4), the solute mass ratio of the protein fiber-oxidized polysaccharide graft product to the ferric chloride solution is preferably 1:1.

[0040] In the step (4), the mass ratio of the ferric chloride to NaBH4 is 5-6:1, preferably 5:1.

[0041] The present application 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 nano-iron complex, which can be used as a nano-iron iron supplement.

[0042] The protein fiber-oxidized polysaccharide iron-loaded complex of the present application can be used for preparing an iron supplement medicine.

[0043] The protein fiber-oxidized polysaccharide iron-loaded complex of the present application can be used for preparing an anti-anemia medicine.

[0044] The application provides a protein fiber-oxidized polysaccharide composite (WPH-DES) and a protein fiber-oxidized polysaccharide iron-carrying composite (abbreviated as WPH-DEX-Fe) carrying iron ions. The protein fiber in the protein fiber-oxidized polysaccharide composite is mixed in equal proportions with hydrolyzed whey protein isolate and unhydrolyzed whey protein isolate, subjected to fiberization treatment, and a protein fiber solution is formed. The protein fiber solution has a large number of free amino groups, and the amino groups and carbonyl groups can undergo Schiff base reaction. In order to make the polysaccharide have more sites to covalently graft with the hydrolyzed protein fiber, the application uses sodium periodate to oxidize glucose to produce the corresponding compound. Then the grafted precise modification of the protein fiber is carried out using the oxidized low molecular weight dextran (dextran 40), and the protein fiber and the oxidized polysaccharide are stably grafted by using the Schiff base reaction to form the protein fiber-oxidized polysaccharide composite, so that the addition of the polysaccharide further enhances the iron chelating capacity. The protein fiber-oxidized polysaccharide iron-carrying composite is reduced to divalent iron ions under the action of sodium borohydride and combined on the protein fiber-oxidized polysaccharide composite, and a protein fiber-oxidized polysaccharide-iron ternary composite is successfully constructed. The iron-carrying composite developed by the application 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 the need of being converted into other forms, greatly improves the iron utilization degree, and after being absorbed, can continue to promote the transport and distribution of iron in the body.

[0045] Compared with the prior art, the application has the beneficial effects that:

[0046] (1) The application uses natural whey protein isolate and polysaccharide as raw materials, has good biocompatibility and low cytotoxicity, and is suitable for long-term use in the human body.

[0047] (2) The application 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, a composite system with high antioxidant property and high iron-carrying capacity is constructed, and the protein fiber with high antioxidant property can effectively stabilize the valence state of divalent iron and reduce the oxidation loss of iron ions during storage and transportation.

[0048] (3) The application uses the crosslinking reaction of the free amino groups of the enzymatically hydrolyzed protein fiber and the carbonyl groups released by the oxidized dextran to form a composite, and the grafting degree of the composite is as high as 86% under the condition of 4°C, which significantly improves the structural stability of the system and enhances the anti-degradation ability of the system under different environmental conditions.

[0049] (4) The protein fiber-oxidized polysaccharide complex constructed by the application has a high iron loading ratio of 1:1, which is significantly higher than that of a traditional iron supplement, and can provide higher iron content at a lower dose, thereby improving the iron supplement effect. Meanwhile, the nanoscale treatment of the complex system ensures the iron carrying capacity and stability, and can meet the market demand for high-concentration iron supplement. The protein fiber-oxidized polysaccharide complex provided by the application can not only meet the requirements of high iron supplement effect and safety in some scenes, but also can be used as an emulsifier, an antioxidant, and a carrier of an anti-anemia drug (such as a functional food, a health food, and a drug development), and has a wider functional expansion and application prospect.

[0050] (5) The iron-loaded complex provided by the application has good biological safety, low cytotoxicity, small intestinal stimulation, and high bioavailability. The cell absorption effect is significantly improved compared with conventional ferrous salts. Compared with ordinary ferrous sulfate, the cell absorption of the protein fiber-oxidized polysaccharide iron-loaded complex is 1.33 times that of ferrous sulfate. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 The figure is for characterization of controllable preparation and binding mechanism of protein fiber-oxidized polysaccharide complex under different reaction times. A is a graph of the grafting degree under different reaction times, B is a comparison graph of the primary and ultimate product contents of the protein fiber-oxidized polysaccharide grafting product, C is a UV full spectrum full band scanning spectrum graph of the protein fiber-oxidized polysaccharide grafting product under different reaction times, and D is an infrared spectrum graph of the protein fiber-oxidized polysaccharide grafting product under different reaction times.

[0052] Figure 2 The figure is for antioxidant capacity detection of the protein fiber-oxidized polysaccharide complex. A is a comparison graph of the reducing power of whey protein fiber after different treatment methods, and B is a comparison graph of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging capacity of whey protein fiber after different treatment methods.

[0053] Figure 3 The figure 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.

[0054] Figure 4 The figure is a transmission electron microscope micrograph of the iron-loaded protein fiber-oxidized polysaccharide complex under different pH and heating conditions.

[0055] Figure 5 The figure is a graph of the promotion effect of the iron-loaded complex of protein fiber after different treatment methods on the growth of Caco-2 cells and a graph of the ferritin content in the iron absorption process. A is a graph of the relative cell survival rate (%) under different concentrations, and B is a graph of the ferritin content (ng / mg) in the cell absorption process.

[0056] Figure 6 Figure 3 shows the iron ion release concentration of protein fiber-loaded iron complex in simulated gastric fluid (SGF) and intestinal fluid (SIF) after treatment with different methods. DETAILED DESCRIPTION

[0057] For a better understanding of the present invention, the present invention is further described below with reference to the following examples. The present invention has many successful embodiments, and six specific embodiments are listed below. However, the scope of protection claimed in the present invention is not limited to these embodiments.

[0058] Example 1: Controllable preparation of protein fiber-oxidized polysaccharide complex and characterization of binding mechanism

[0059] Sample preparation: (1) The whey protein isolate dispersion (4% weight-to-volume ratio) was adjusted to pH = 8 by adding alkali, heated in a 100℃ water bath for 10 min, and then alkaline protease (enzyme mass dosage is 4% of protein mass) was added and heated at 50℃ for 150 min, and then centrifuged. The supernatant was taken and freeze-dried to form whey protein isolate hydrolyzed powder. The whey protein isolate hydrolyzed powder and whey protein isolate powder were mixed in a mass ratio of 1:1, dissolved in water to prepare a dispersion with a concentration of 20 mg / ml, acid was added to adjust the pH to 2, and hydrated at 4℃ overnight. Then, it was heated in a 90℃ water bath for 5 h, quickly transferred to an ice water bath for cooling, and cooled to room temperature to terminate the self-assembly of the microstructure to obtain a hydrolyzed protein fiber solution, which was recorded as WPH.

[0060] (2) Dextran (dextran 40) and sodium periodate were dissolved in 20 mM acetate buffer to a dextran concentration of 2.5 mg / ml and a sodium periodate concentration of 10 mM. The mixture was stirred on a magnetic stirrer for 90 min in the dark, and then ethylene glycol was added to terminate the reaction (3.73 µl of ethylene glycol was added per 1 mg of sodium periodate). The mixture was then dialyzed against 0.15 M sodium chloride solution (twice for 1 h each) and pH 7.4 phosphate buffer (once for 3 h each), followed by overnight dialyzation against water to obtain an oxidized dextran solution.

[0061] (3) Oxidized dextran solution, hydrolyzed protein fiber solution and sodium cyanoborohydride (NaBH3CN) were mixed at a solute mass ratio of 1:1:1, stirred evenly, and reacted at 4°C for 2, 3, 4, 5, 6, 7, and 12 h, respectively, to obtain protein fiber-oxidized polysaccharide grafted products at different reaction times, which were recorded as WPH-DEX.

[0062] (4) The protein fiber-oxidized polysaccharide grafting product is mixed with FeCl3 6H2O at a ratio of 1:1 of solute (the mass of the ferric chloride hexahydrate is denoted as solute) to solute, stirred at room temperature for 0.5 h, then slowly added with NaBH4, and the mass ratio of sodium borohydride to ferric chloride hexahydrate is 1:5, and it is allowed to stand for 1 h, and after stabilization, it is dialyzed with pure water overnight to prepare a protein fiber-oxidized polysaccharide composite iron-loaded system, denoted as WPH-DEX-Fe.

[0063] The control group: whey protein isolate is dissolved in water to prepare a dispersion with a concentration of 20 mg / ml, and the pH is adjusted to 2 with acid, and hydrated overnight at 4°C. Then heated in a water bath at 90°C for 5 h, quickly transferred to an ice water bath for cooling, cooled to room temperature to terminate the self-assembly of the microstructure, to prepare a whey protein isolate fiber solution, denoted as WPI.

[0064] According to the above step (3), a whey protein isolate fiber-oxidized polysaccharide grafting product can be prepared, denoted as WPI-DEX.

[0065] The grafting reaction of protein fibers and oxidized polysaccharides under different reaction times is shown in the following figure Figure 1 , wherein Figure A is a graph of the grafting degree under different reaction times, 4 mL of OPA reagent is added to 200 μL of sample and mixed thoroughly, and incubated at 35°C for 2 min. 200 μL of ultrapure water is used instead of the sample as a control, and the absorbance is measured at 340 nm. Lysine is used as a standard to draw a standard curve, and the free amino acid content is calculated. The calculation formula of the grafting degree (DG) is as follows: , and the highest grafting degree is 86% at 6 h. It shows that the synergistic modification method of the present application not only enhances the structural stability of the composite system, but also improves its anti-degradation ability under different environmental conditions.

[0066] Figure B is a comparison graph of the primary and final product contents of the protein fiber-oxidized polysaccharide grafting product. The sample is diluted with 0.1% sodium dodecyl sulfate to a protein concentration of 2 mg / mL, and the absorbance at 294 nm and 420 nm is measured using a UV spectrophotometer, and is denoted as A 294 and A 420The primary products include incompletely reacted proteins and polysaccharides, and the initially formed protein polysaccharide graft copolymer. The final product is a brownish black melanoid substance, and the test results show that the reaction has been controlled in the primary stage. The high content of the primary product shows that the synergistic modification method of the present application has controlled the Maillard reaction in the primary stage, and the early grafting can reduce the interference to the active sites of the protein, thereby enhancing the stability and emulsifying properties of the protein polysaccharide system. In addition, the test results show that the protein fiber-oxidized polysaccharide has a high grafting degree at 4℃, and the grafting degree is the highest when the reaction is performed for 6 h. The high grafting degree can improve the stability of the system. Therefore, the present application preferably has a reaction time of 6 h. The protein fiber-oxidized polysaccharide graft product prepared by reacting for 6 h is used as the raw material in the subsequent experiments, and is denoted as WPH-DEX.

[0067] C is a UV full spectrum full wave band scanning spectrum of the protein fiber-oxidized polysaccharide graft product under different reaction times. As shown in FIG. C, the WPI has two main absorption peaks in the wavelength range of 220~500 nm. The first absorption peak is located at 220~240 nm, which is caused by the characteristic UV absorption of the peptide bond. With the progress of the grafting reaction, the peak intensity decreases and red shifts, which is presumably due to the formation of Schiff base and other structures by the reaction of the reducing end carbonyl group of the polysaccharide and the protein. The second absorption peak is in the range of 260~320 nm, and the intensity increases, but the maximum absorption wavelength does not change, which is presumably due to the exposure of the aromatic amino acid residues in WPH caused by the dextran grafting, thereby enhancing the UV absorption. The red shift and intensity change of the absorption peak indicate that the tertiary structure of the protein is changed, which shows that the polysaccharide (dextran) in the composite system prepared by the present application is successfully grafted onto the protein fiber.

[0068] D is an infrared spectrum of the protein fiber-oxidized polysaccharide graft product under 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 the pure WPI fiber, which may be due to the decrease in the content of the carbonyl group and the gradual polymerization into larger molecules. However, the Schiff base in the reaction product has an absorption peak at this position, resulting in some absorption at a wave number of 1654 cm -1 . At the same time, the absorption peak of the amide II band (N-H bending vibration, 1529 cm -1 ) is significantly weakened, indicating that a large number of carbonyl and amino groups between the dextran and the WPI fiber are lost, and the carbonyl-amino reaction occurs. The amide I band (1645 cm -1 ) and the amide III band (1240 cm -1The significant change after the dextran grafting indicates that the tertiary structure of the protein has been changed by the polysaccharide. The complex system prepared by the present application proves the high grafting efficiency of dextran to the protein fiber, and the high grafting degree not only enhances the structural stability of the complex system, but also improves the anti-degradation ability of the complex system under different environmental conditions.

[0069] Example 2: Detection of the antioxidant capacity of the protein fiber-oxidized polysaccharide complex

[0070] The comparison chart of the reducing power and free radical scavenging capacity of the whey protein isolate fibers WPI, WPH and WPH-DEX treated by different methods is shown in FIG. 1, wherein FIG. 1A is the reducing power, and FIG. 1B is the DPPH free radical scavenging capacity. Figure 2

[0071] The determination of the reducing power is as follows: 50 μL of the protein dispersion (WPI, WPH or WPH-DEX) is taken to an enzyme-labeled plate, FeCl3·6H2O (0.1 M) is added so that the mass ratio of Fe to protein is 1:5, then an excess of 1,10-phenanthroline is added, and the absorbance at 512 nm is measured after 15 min, which is OD 512 nm. The determination of the DPPH scavenging capacity is as follows: a 0.1 mmol / l DPPH methanol solution is prepared and stored in the dark. The protein fiber solution treated by different methods is mixed with the DPPH methanol solution, and is placed in the dark for 1 h. The absorbance of the mixture at 517 nm is immediately measured and is marked as A1. At the same time, an equal volume of distilled water is mixed with the DPPH-methanol solution, and the absorbance A2 is measured as a blank control. An equal volume of the sample solution is mixed with the methanol solution, and the absorbance A3 is measured as a control. The free radical scavenging rate is calculated using the following formula:

[0072]

[0073] When the reducing power and DPPH free radical scavenging capacity of the protein fibers after different treatments are analyzed, it is observed that the reducing power and scavenging capacity of the protein fiber samples treated by enzyme hydrolysis and polysaccharide grafting are significantly better than those of the pure protein fiber. It is proved that the complex prepared by the present application has high antioxidant capacity, which is helpful to stabilize the divalent iron ion.

[0074] Example 3: Detection of the divalent iron content in the protein fiber-oxidized polysaccharide iron-loaded complex

[0075] ​The whey protein isolate fiber solution was mixed with FeCl3·6H2O at a ratio of 1:1 (mass of the solute, i.e., the mass of the iron chloride hexahydrate, to the mass of the solvent), stirred at room temperature for 0.5 h, then slowly added NaBH4, with a mass ratio of sodium borohydride to iron of 1:5, and allowed to stand for 1 h. After stabilization, the whey protein isolate fiber-iron complex, denoted as WPI-Fe, was prepared by dialysis overnight using purified water.

[0076] The whey protein isolate fiber solution was replaced with the hydrolyzed protein fiber solution prepared in step (1) of Example 1 to prepare a hydrolyzed whey protein fiber-iron complex, denoted as WPH-Fe.

[0077] The 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 were subjected to X-ray photoelectron spectroscopy (XPS) analysis to study their composition, and the results are shown in FIG. 3. Figure 3 The XPS identified the composition of iron, distinguishing between the iron core and the outer layer of the nanoparticles. In this experiment, the relative content of iron (II) and iron (III) in the samples was analyzed to demonstrate that the samples had enhanced antioxidant capacity, stability of divalent iron, and loading capacity for divalent iron under the conditions of synergistic modification. Through XPS analysis, it was determined that the peak area ratio of iron (II) to iron (III) in the three samples was 23:77, 49:51, and 77:23, respectively. This indicates that the protein fiber-oxidized polysaccharide iron-loaded complex treated by synergistic modification has higher antioxidant activity and can stabilize the valence state of iron, thereby promoting the absorption and utilization of iron in the gastrointestinal tract.

[0078] Example 4: Characterization of the iron transport stability of the protein fiber-oxidized polysaccharide complex

[0079] As shown in FIG. 4, the protein fiber-oxidized dextran iron-loaded complex was observed under a transmission electron microscope (TEM). From the figure, it can be clearly observed that as the pH value increased from 3 to 9, the dispersion state of the protein fibers changed significantly, showing a trend from dispersion to aggregation. Figure 4 At a lower pH value (e.g., pH = 3), the protein fiber-oxidized dextran iron-loaded complex exhibited a relatively clear nanofiber structure, with uniform distribution between the fibers and no obvious aggregation phenomenon. This may be due to the fact that under acidic conditions, the charge state on the surface of the protein fibers helps to maintain the electrostatic repulsion between the fibers, thereby maintaining the dispersion stability of the fibers.

[0080]

[0081] ​As the pH value gradually increased to neutral or alkaline conditions (e.g., pH = 7 and 9), it was observed that the protein fibers began to exhibit obvious aggregation. This aggregation may be due to the reduction of the charge on the surface of the protein fibers under neutral conditions, resulting in a decrease in electrostatic repulsion and an increase in attractive forces between fibers, thus promoting the aggregation of the fibers.

[0082] When the pH value was further increased to 9, the aggregation of the protein fibers was more severe, forming larger aggregates. Under alkaline conditions, the protein fibers may have undergone structural changes, such as the beta-sheet structure may have transformed into other forms, further promoting the aggregation of the fibers. In addition, metal ions may have stronger interactions with the protein fibers and polysaccharides at higher pH values, which may also promote the aggregation of the fibers.

[0083] After heating the protein fiber-oxidized polysaccharide iron-loaded complex (product pH = 4-5) at 90°C for two hours, the sample still maintained the fiber morphology without obvious changes, indicating that the sample performed well in terms of thermal stability, providing stable conditions for subsequent processing.

[0084] It is shown that even under extreme pH conditions and high-temperature heating, the protein fibers after the synergistic modification strategy still maintain the fiber morphology, indicating that the protein fiber iron-loaded complex has higher stability.

[0085] Example 5: Bioavailability determination of protein fiber-oxidized polysaccharide iron-loaded complex

[0086] The promotion effect of protein fiber iron-loaded complex treated by different methods on Caco-2 cell growth and the iron protein content in the cell absorption process are shown in FIGS. Figure 5 Figure 5 -A is the relative cell survival rate (%) at different concentrations, Figure 5 -B is the iron protein content (ng / mg) in the cell absorption process. Through Caco-2 cell toxicity and cell absorption verification, it is shown that the iron-loaded system is non-toxic, and has a significant effect on cell absorption of iron.

[0087] Cell toxicity evaluation: standard CCK-8 experiment was used to measure the cytotoxicity of Caco-2 cells. The CCK-8 experiment steps are as follows: 5.0 × 10 3 cells were inoculated in each well of a 96-well plate and cultured at 37°C, 5% CO2 for 24 h. The cell culture medium was removed, and the sample diluted with culture medium was added to each well. Then the cells were cultured for another 24 h in an environment containing 5% CO2. 10 ul of CCK-8 solution was added to each well. After incubation in the incubator for an appropriate time, the absorbance at 450 nm was measured using a microplate reader to determine the cell survival rate. ​

[0088] Cell uptake evaluation, once the complete Caco-2 cell monolayer membrane is formed, can be used to evaluate the bioavailability of iron. First, the original culture medium in the culture plate is discarded, and then 100 ul of the freshly prepared sample solution is added to each well of the six-well plate. Subsequently, 600 ul of DMEM medium is added, and three repeated wells are set up for each group. Then the cells are cultured in the cell incubator for 24 h. After the culture medium is discarded, the upper chamber is washed twice with pre-cooled PBS. An appropriate amount of RIPA lysis buffer is added on ice to collect the cell lysate. The protein content is measured 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.

[0089] Figure 5 The results show that all the samples prepared by the present application have a positive effect on the growth and division of Caco-2 cells at this concentration, but as the concentration increases, only the dextran grafted protein fiber has a cell growth of more than 100% at 0.5 mg / ml, proving that at a higher concentration, the protein fiber-oxidized polysaccharide iron-loaded complex provided by the present application has no toxic effect on cell growth.

[0090] Figure 5 The results show that the protein fiber-oxidized polysaccharide iron-loaded complex has a significant promoting effect on cell growth, and after Caco-2 cell uptake, the ferritin content of the grafted polysaccharide protein fiber iron is much higher than that of the ungrafted protein fiber, which is 1.33 times that of the conventional iron supplement ferrous sulfate, proving that the protein fiber-oxidized polysaccharide iron-loaded complex has better bioavailability.

[0091] Example 6: Evaluation of the Digestive Capacity of Protein Fiber-Oxidized Polysaccharide Iron-Loaded Complex in the In Vitro Gastrointestinal Tract

[0092] The iron ion release concentration diagram of the protein fiber iron-loaded complex WPI-Fe, WPH-Fe and WPH-DEX-Fe treated by different methods in simulated gastric fluid (SGF) and intestinal fluid (SIF) is shown in Figure 6 The simulated gastric fluid (SGF) is prepared by weighing 0.2 g of NaCl, dissolving it in 100 ml of water, adjusting the pH to 2.0 with 1 mol / l HCl, and adding an appropriate amount of pepsin (enzyme concentration of 10 mg / ml).

[0093] Simulated gastric digestion: A solution of protein fiber iron complex at a concentration of 2.5 mg / ml was prepared. 20 ml of the solution was taken into a conical flask, and the pH was adjusted to 2.0 with 1 mol / l HCl. Then the flask was heated to 37℃ for 10 min. Subsequently, 20 ml of simulated gastric fluid (SGF) was added, shaken well, sealed, and shaken on a shaker at 37℃ for 30 min, 60 min, 90 min, and 120 min, respectively. After the reaction was completed, the Fe 2+ content in the supernatant was determined.

[0094] Simulated intestinal fluid (SIF): 0.68 g of KH2PO4 and 0.062 g of NaOH were weighed and dissolved in 100 ml of water, and then the pH was adjusted to 6.8 with 1 mol / l NaOH. An appropriate amount of trypsin was added, and the enzyme concentration was 10 mg / ml. For the simulated intestinal digestion process, after the simulated gastric digestion was completed, the pH was adjusted to 6.8 with 1 mol / l NaOH, and preheated at 37℃ for 5 min. Then 4 ml of simulated intestinal fluid (SIF) was added, shaken well, sealed, and shaken on a shaker at 37℃ for 30 min, 60 min, 90 min, and 120 min, respectively. After the reaction was completed, the reaction mixture was quickly heated to 90℃ to inactivate the enzyme, and kept for 10 min. After cooling to room temperature, the Fe 2+ content in the supernatant was determined.

[0095] Figure 6 The results show that the WPH-DEX complex increases the content of divalent iron in simulated gastric fluid (pH=2.0). Due to the hydrolysis of protein fibers by pepsin, the stability of the peptide-iron chelate is destroyed, and the fibers are temporarily dispersed. At the same time, the strong acidic environment leads to a decrease in the binding ability between iron ions and fibers, causing the dissociation of iron ions from the fibers, resulting in the temporary presence of free divalent iron ions. In comparison, WPI and WPH have poor stability in the complex system, low divalent iron content, and cannot stably release. After entering the simulated intestinal fluid, the solubility of iron ions in the neutral or alkaline environment decreases, and they are more easily recombined with the fibers. Therefore, the divalent iron ions and the fibers recombine, and the stable peptide-iron complex is reformed in the neutral environment, reducing the free divalent iron ions, improving the bioavailability, and reducing the stimulation of the gastrointestinal tract. At the same time, the fibers have good antioxidant properties, protecting the divalent iron from oxidation, which is conducive to intestinal absorption. It shows that the new complex system prepared by the application can make the protein fibers and iron ions recombine in the intestine to form iron-loaded complexes, which can be subsequently absorbed by the intestine, improve the bioavailability, and also reduce the damage and stimulation of free divalent iron ions to the human gastrointestinal tract.

[0096] The foregoing examples are to be construed as merely illustrative of the presently disclosed embodiments, and do not exhaust the scope of the present application. Furthermore, various modifications to both the examples listed herein and the methods and compositions of the application will be apparent to those skilled in the art, and this application is intended to encompass such modifications within the scope and spirit of the application. Although the application has been described in conjunction with specific preferred embodiments thereof, it will be understood that the application is not limited to these embodiments. In fact, various modifications to the described embodiments will be apparent to those skilled in the art, and the general principles defined herein can be applied to other embodiments. Therefore, to the extent that modifications of the described embodiments incorporating some but not other features of the application are within the spirit and scope of the application, they should be and are intended to be included within the scope of the application.

Claims

1. A method for preparing a protein fiber-oxidized polysaccharide complex, characterized by The method comprises the following steps: (1) whey protein isolate is dissolved in deionized water to prepare a whey protein isolate solution with a concentration of 3-4% by weight by volume, the pH is adjusted to 8, and the solution is heated to 95-100°C for 10-15 min, then alkaline protease is added, and the solution is heated at 50-55°C for 2-4 h, the supernatant is obtained by centrifugation, and the whey protein isolate is freeze-dried to obtain a whey protein isolate enzymolysis powder; the whey protein isolate and the whey protein isolate enzymolysis powder are mixed at a mass ratio of 1:1 to prepare a dispersion solution with a concentration of 20-25 mg / mL, the pH is adjusted to 1.5-2 by adding acid, the solution is hydrated at 0-4°C for 8-15 h, and then the solution is heated and stirred at 85-90°C for 4-8 h to prepare a hydrolyzed protein fiber solution; (2) dextran is subjected to oxidation reaction under the action of sodium periodate to prepare an oxidized dextran solution; (3) the oxidized dextran solution and the hydrolyzed protein fiber solution are subjected to grafting under the action of sodium cyanoborohydride at 0-4°C to prepare a protein fiber-oxidized polysaccharide composite.

2. The method for preparing the protein fiber-oxidized polysaccharide composite according to claim 1, characterized in that the reaction step of step (2) is as follows: dextran and sodium periodate are dissolved in an acetic acid buffer to prepare a solution with a dextran concentration of 2-3 mg / mL and a sodium periodate concentration of 10-15 mM; the solution is stirred in the dark for 1-2 h, then ethylene glycol is added to terminate the reaction, and small molecules are removed by dialysis to prepare an oxidized dextran solution; the reaction step of step (3) is as follows: the oxidized dextran solution, the hydrolyzed protein fiber solution and sodium cyanoborohydride are mixed at a solute mass ratio of 1:0.8-1.2:0.8-1.2, uniformly stirred, and reacted at 0-4°C for 5-8 h to prepare a protein fiber-oxidized polysaccharide composite.

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

4. The use of the protein fiber-oxidized polysaccharide composite according to claim 3 in the preparation of a carrier for active substances that are prone to oxidation.

5. The use of the protein fiber-oxidized polysaccharide composite according to claim 3 in the preparation of an anti-anemia drug carrier. The method comprises the following steps:

6. A method for preparing a protein fiber-oxidized polysaccharide iron complex, characterized by (1) whey protein isolate is dissolved in deionized water to prepare a whey protein isolate solution with a concentration of 3-4% by weight by volume, the pH is adjusted to 8, and the solution is heated to 95-100°C for 10-15 min, then alkaline protease is added, and the solution is heated at 50-55°C for 2-4 h, the supernatant is obtained by centrifugation, and the whey protein isolate is freeze-dried to obtain a whey protein isolate enzymolysis powder; the whey protein isolate and the whey protein isolate enzymolysis powder are mixed at a mass ratio of 1:1 to prepare a dispersion solution with a concentration of 20-25 mg / mL, the pH is adjusted to 1.5-2 by adding acid, the solution is hydrated at 0-4°C for 8-15 h, and then the solution is heated and stirred at 85-90°C for 4-8 h to prepare a hydrolyzed protein fiber solution; (2) dextran is subjected to oxidation reaction under the action of sodium periodate to prepare an oxidized dextran solution; (3) the oxidized dextran solution and the hydrolyzed protein fiber solution are subjected to grafting under the action of sodium cyanoborohydride at 0-4°C to prepare a protein fiber-oxidized polysaccharide composite. (3) oxidized dextran solution, hydrolyzed protein fiber solution is grafted under the action of sodium cyanoborohydride at 0-4℃ to prepare protein fiber-oxidized polysaccharide composite; (4) the protein fiber-oxidized polysaccharide composite is loaded with ferric chloride and reduced by NaBH4 to prepare protein fiber-oxidized polysaccharide iron-loaded composite.

7. The method of claim 6, wherein The reaction step of step (4) is: The protein fiber-oxidized polysaccharide graft product is mixed with a ferric chloride hexahydrate solution at a solute mass ratio of 1:0.8-1.2, stirred at room temperature for 20-50 min, then slowly added with NaBH4, placed for 1-2 h, purified by water dialysis to prepare the protein fiber-oxidized polysaccharide iron-loaded composite.

8. The protein fiber-oxidized polysaccharide iron-loaded composite prepared by the method of claim 6 or 7.

9. The use of the protein fiber-oxidized polysaccharide iron-loaded composite of claim 8 in the preparation of an iron supplement.

10. The use of the protein fiber-oxidized polysaccharide iron-loaded composite of claim 8 in the preparation of an iron supplement drug or an anti-anemia drug.

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