Whey protein fibril loaded nano iron oxide and preparation method thereof

Abstract

The preparation method comprises the following steps: placing a whey protein isolate solution at the temperature of 0-4 DEG C for hydration, adjusting the pH value to be acidic, heating and stirring for reaction, and cooling to obtain a whey protein fibril solution; the preparation method comprises the following steps: adding a bivalent iron salt and a trivalent iron salt into a whey protein fibril solution, adjusting the pH value to be alkaline, heating to 55-60 DEG C, and reacting to obtain the whey protein fibril loaded nano-iron oxide. The whey protein fibril loaded nano-iron oxide disclosed by the invention is high in iron loading capacity, good in stability, good in digestion resistance, improved in oxidation resistance and good in biological safety and bioavailability; compared with conventional ferrite, the cell absorption iron is remarkably improved.

Description

Technical Field

[0001] The present invention relates to a whey protein nanofiber loaded with nano iron oxide and a preparation method thereof. Background Art

[0002] Iron is an important element indispensable to the human body and participates in functions such as energy metabolism, oxygen binding, DNA biosynthesis and repair in organisms. Iron deficiency anemia is a major global public health problem and is prone to occur in children, adolescent women and women of childbearing age, attracting wide attention and emphasis in society. Oral iron supplements are commonly used to prevent and treat iron deficiency anemia, but they usually cause side effects and damage to the body, such as diarrhea, upper abdominal pain and constipation.

[0003] Common iron supplements can be divided into three categories. Inorganic iron salts such as ferrous sulfate have a high biological utilization rate, but have a relatively unpleasant rust smell, are easy to oxidize, have active chemical properties, and have adverse color reactions with substances such as polyphenols, phytic acid, flavonoids, and calcium, resulting in flavor deterioration. Organic acid irons such as ferrous fumarate, etc., are absorbed in the form of ions. However, organic acid irons are prone to oxidation during preparation and storage and can cause gastrointestinal discomfort. Macromolecular complex iron supplements such as polysaccharide iron, etc., can effectively reduce the irritation to the gastrointestinal mucosa and have good stability, but their iron supplementation effect is lower than that of the first two types of iron supplements. Therefore, the development and research of new iron fortifiers are the key to solving the difficulties and challenges.

[0004] Iron oxide nanoparticles are widely used in the biomedical field and food pigments due to their low toxicity, superparamagnetism and simple preparation methods. Iron oxide nanoparticles are considered suitable for iron fortification or iron supplementation in foods, and have fewer side effects on the stomach and intestines compared to ferrous sulfate, causing fewer changes in the taste and texture of foods. Existing research uses chitosan and folic acid to coat iron oxide nanoparticles and finds that folic acid-coated iron oxide nanoparticles have certain potential in the treatment of iron deficiency anemia. However, people have ignored the fact that proteins have strong stability, good carrying ability and high biocompatibility. At the same time, whey protein has the function of promoting cell iron absorption, which indicates that proteins may become potential carriers of iron oxides.

[0005] Whey protein nanofibers are formed by heating whey protein isolate at a pH significantly lower than its isoelectric point. Amyloid fibers have a nanoscale thickness and a microscale contour length, and this excellent aspect ratio endows amyloid fibers with good gel-forming ability and the ability to form stable emulsions. Compared with natural proteins, amyloid fibers show stronger resistance under acidic and thermal conditions, providing greater stability to the systems they bind to.

[0006] There has been little research on carriers of iron oxide nanoparticles in the prior art. It is necessary to develop a new loading system for iron oxides to improve the transport efficiency and bioavailability of iron oxides and provide new iron supplements. Summary of the Invention

[0007] Aiming at the problems of poor taste, strong irritation, poor stability or poor iron supplement effect of iron supplements in the prior art, the present invention provides a method for preparing whey protein nanofibers loaded with nano-iron oxides. Iron oxides are in-situ synthesized on the nano-protein nanofibers by the co-precipitation method, forming a stable colloidal system, avoiding the problem of protein denaturation at high temperature (160 °C) in the normal synthesis method of iron oxides. The obtained whey protein nanofibers can be used as a composite iron supplement, having good stability, biosafety and bioavailability.

[0008] The technical solution adopted by the present invention is as follows: A method for preparing whey protein nanofibers loaded with nano-iron oxides, the method comprising: (1) A whey protein isolate solution is placed at 0-4 °C for hydration, the pH value is adjusted to be acidic, heated and stirred for reaction, and then cooled to obtain a whey protein nanofiber solution; (2) Ferrous salts and ferric salts are added to the whey protein nanofiber solution, the pH value is adjusted to be alkaline, and heated to 55-60 °C for reaction to obtain whey protein nanofibers loaded with nano-iron oxides; In the step (1), the weight-volume concentration of the whey protein isolate solution is preferably 2-3%.

[0009] The hydration time is 8-15 hours.

[0010] In the step (1), after hydration, an acid is added to adjust the pH value to 1.5-2.

[0011] Further, in the step (1), when adjusting the pH value with an acid, generally 3-6 M hydrochloric acid is added.

[0012] In the step (1), generally the reaction is carried out at a temperature of 85-90 °C. The reaction time is preferably 5-8 hours.

[0013] In the step (1), 0.01% (weight / volume) of sodium azide can be added to the obtained whey protein nanofiber solution to inhibit the growth of microorganisms.

[0014] In the step (2), the ferrous salts and ferric salts are both soluble iron salts, which can be sulfates, nitrates or chlorides.

[0015] Preferably, the ferrous salt is ferrous chloride tetrahydrate, and preferably the ferric salt is ferric chloride hexahydrate.

[0016] More preferably, the molar ratio of the ferrous salt to the ferric salt is preferably 0.4 to 1.4:1.

[0017] Preferably, the mass ratio of ferrous chloride tetrahydrate to ferric chloride hexahydrate is 1:1 to 3.

[0018] In the step (2), the total mass of the ferrous salt and the ferric salt and the mass of the solute of the whey protein fibril solution are in a ratio of 1:2 to 3.

[0019] In the step (2), ammonia water is generally added dropwise to adjust the pH value. Further, the pH value is adjusted to 9 to 10 by adding dropwise ammonia water.

[0020] The mass concentration of ammonia water is generally 25% - 30%.

[0021] In the step (2), the heating reaction time is generally 40 to 60 minutes.

[0022] Further, the step (1) preferably comprises the following steps: Dissolve whey protein isolate in deionized water under stirring to obtain a whey protein isolate solution with a weight - volume concentration of 2% - 3%, then place it at 0 - 4 °C for 8 - 15 hours for hydration, then add acid to adjust the pH value to 1.5 - 2, heat and stir - react at 85 - 90 °C for 5 - 8 hours, and cool to obtain a whey protein fibril solution; Further, the step (2) preferably comprises the following steps: Add the ferrous salt and the ferric salt to the whey protein fibril solution, add dropwise ammonia water under stirring, adjust the pH value to 9 - 10, then heat to 55 - 60 °C and react for 40 - 60 minutes, and dialyze to remove the excess ammonia to obtain whey protein fibril - loaded nano - iron oxides.

[0023] The present invention also provides the whey protein fibril - loaded nano - iron oxides prepared by the above method, and the application of the whey protein fibril - loaded nano - iron oxides as an iron supplement.

[0024] Further, the whey protein fibril - loaded nano - iron oxides of the present invention can be used for preparing iron - supplement drugs.

[0025] The whey protein fibril - loaded nano - iron oxides of the present invention can be used for preparing anti - anemia drugs.

[0026] In the step (1), hydration can increase the solubility and dispersibility of proteins, thus helping protein molecules to better unfold and interact in subsequent processing, and form a stable protein fibril structure. Hydration at low temperature can also avoid the thermal denaturation of proteins and maintain the natural structure and function of proteins.

[0027] In the previous work of the present invention, it was observed that although whey protein isolate amyloid fibrils have limited chelating ability for iron ions, they show good ability to load metal nanoparticles and can improve their dispersibility and stability.

[0028] In the present invention, iron oxides were in-situ synthesized on nano-whey protein fibrils by co-precipitation method to form a stable colloidal system, avoiding the problem of protein denaturation at high temperature in the normal synthesis method of iron oxides. The present invention also explored the synthesis of iron oxides with different ratios of Fe(III) and Fe(II) and studied the effect of valence change on bioavailability. The structure of the composite iron supplement was studied, and in vitro digestion release and cell experiments were carried out to explore the release rate, secondary structure and bioavailability of iron ions in the composite system.

[0029] The results show that the composite iron supplement of whey protein fibril loaded with nano-iron oxides of the present invention has good stability, biosafety and bioavailability.

[0030] The beneficial effects of the present invention are as follows: (1) Iron oxides were in-situ synthesized on nano-protein fibrils by co-precipitation method, and the reaction temperature was below 60 °C, effectively avoiding protein denaturation. Whey protein isolate fibrils can effectively transport iron oxide nanoparticles and form a stable colloidal system.

[0031] (2) The whey protein fibril loaded with nano-iron oxides has good stability after gastrointestinal digestion, showing good anti-digestion ability. Only 10% of the iron oxide nanoparticles dissolve during the digestion process in simulated gastric juice, and the rest remain in the form of iron oxide nanoparticles and can be directly absorbed by the intestine.

[0032] (3) After being treated with whey protein fibril-iron oxide nanoparticles for 24 h, it did not produce toxic effects on HT-29 and Caco-2 cells, indicating that the whey protein fibril loaded with nano-iron oxides of the present invention has good biosafety as an iron supplement.

[0033] (4) Compared with FeSO4, the absorption rate of iron in the composite iron supplement by Caco-2 cells increased by 1.3 times. The whey protein fibril loaded with nano-iron oxides of the present invention has better bioavailability. Description of the Drawings

[0034] Figure 1 is the TEM photograph of the composite of whey protein fibrils and whey protein fibrils loaded with nano-iron oxides, where Figure 1 the left figure is the whey protein fibril, the middle figure is Fe1-WPF, and the right figure is Fe2-WPF.

[0035] Figure 2 In vitro digestion iron ion release concentration diagrams of complexes of whey protein nanofibers loaded with nano iron oxides prepared by different methods.

[0036] Figure 3 Diagrams of changes in microscopic morphology during in vitro digestion of whey protein nanofibers and complexes of whey protein nanofibers loaded with nano iron oxides.

[0037] Figure 4 Diagrams of the cytotoxicity and cellular uptake of complexes of whey protein nanofibers loaded with nano iron oxides prepared by different methods on Caco-2 cells. The left diagram is the cell viability diagram, and the right diagram is the ferritin content (ng / mg) diagram of Caco-2 cells during cellular uptake. Detailed implementation manners

[0038] The technical solutions of the present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0039] Example 1 (1) Preparation of whey protein nanofibers, the steps are as follows: First, dissolve whey protein isolate powder in deionized water under continuous stirring to obtain a 2% (weight / volume) whey protein isolate solution, and then hydrate overnight at 4°C. The fully hydrated whey protein isolate was quickly adjusted to pH 2.0 with 6M hydrochloric acid, and then heated in a water bath at 90°C for 6 hours while continuously stirring. After that, the stock solution containing fibers was cooled in an ice-water mixture, and 0.01% (weight / volume) sodium azide was added to inhibit microbial growth. The prepared whey protein nanofibers are denoted as WPF.

[0040] (2) FeCl2·4H2O and FeCl3·6H2O were dissolved in an aqueous solution containing 1 g of whey protein nanofibers at a mass ratio of 3:1, 2:1, 1:1, 1:2, and 1:3. The total mass of FeCl2·4H2O and FeCl3·6H2O was kept at 0.5 g, and the mixture was stirred with a magnetic stirrer. The pH value of the solution was titrated to 10 with 25 wt% ammonia water. Subsequently, the solution was heated in a water bath at 60°C for 50 minutes. Once the reaction was fully completed, excess ammonia was removed by dialysis through a dialysis bag with a molecular weight of 8000 - 14000 for 48 hours to obtain a complex of whey protein nanofibers loaded with nano iron oxides.

[0041] The mass ratios of FeCl2·4H2O and FeCl3·6H2O of the samples with good stability screened out were 1:1 and 3:1 respectively, denoted as Fe1-WPF and Fe2-WPF.

[0042] Transmission electron microscopy (TEM) images of native whey protein fibrils and nanoscale iron oxide-loaded native whey protein fibril composites are as follows Figure 1 shown. Among them Figure 1 the left figure is native whey protein fibrils, the middle figure is Fe1-WPF, and the right figure is Fe2-WPF.

[0043] Figure 1 It shows that native whey protein fibrils have good fibrillar morphology, and the prepared fiber composite system combined with iron oxide still maintains good fiber shape, and particles of about 10 nm in size are combined on the fibers.

[0044] The in vitro digestion experiment was carried out on the nanoscale iron oxide-loaded native whey protein fibril composite. Simulated gastric digestive juice (2 mg / mL NaCl, 10 mg / mL pepsin, pH 2) and simulated intestinal digestive juice (5 mM KH2PO4, 0.62 mg / mL NaOH, pH 6.8, 10 mg / mL trypsin) were prepared. The sample was diluted to 4 mg / mL and the pH of the solution was adjusted to 2.0 with 1 M hydrochloric acid, and preheated at 37 °C for 10 minutes, then mixed with the same volume of simulated gastric juice and reacted with stirring for 120 minutes. After the simulated gastric digestion was completed, the gastric digest was adjusted to pH 6.8 with 1 M NaOH, preheated to 37 °C for 5 minutes, then mixed with the simulated intestinal digestive juice, shaken well, and reacted with stirring at 37 °C. During this period, samples were taken every 30 minutes to determine the iron content by o-phenanthroline ultraviolet-visible spectrophotometry.

[0045] After digestion in simulated gastric juice for 2 h and in simulated intestinal juice for 2 h, the iron ion concentrations of the nanoscale iron oxide-loaded native whey protein fibril composites are as follows Figure 2 shown. The results show that only part of the iron oxide is dissolved. Among them, 10% of the iron oxide in Fe1-WPF is dissolved during digestion, while 30% of Fe2-WPF is dissolved, and Fe1-WPF shows stronger resistance to digestion.

[0046] Transmission electron microscopy (TEM) images of native whey protein fibrils and nanoscale iron oxide-loaded native whey protein fibril composites during digestion are as follows Figure 3 shown. Among them Figure 3 Figure A is the simulated gastric digest of native whey protein fibrils, Figure B is the simulated gastric digest of Fe1-WPF, Figure C is the simulated gastric digest of Fe2-WPF, Figure D is the simulated intestinal digest of native whey protein fibrils, Figure E is the simulated intestinal digest of Fe1-WPF, and Figure F is the simulated intestinal digest of Fe2-WPF.

[0047] The results showed that after digestion by gastric juice, the structure of whey protein fibrils was not destroyed and still maintained a good fibrillar structure. After digestion by intestinal juice, the fibrils were digested into short rod shapes, and the WPF loaded with iron oxide showed some aggregation and could not maintain the short rod-shaped fibrillar structure. This indicates that in the intestine, whey protein fibrils are digested into smaller peptides, which is beneficial to the release and absorption of iron oxide.

[0048] Cytotoxicity experiment. Cytotoxicity was measured by a standard CCK-8 assay using Caco-2 and HT-29 cell lines. The CCK-8 assay procedure was 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 then the samples that had been digested and diluted with the 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 incubation in the incubator for 2 h, the absorbance at 450 nm was measured using a microplate reader to determine the cell viability.

[0049] Cell uptake experiment. Caco-2 cells were seeded into a six-well plate at a density of 5×10 4 cells per well and cultured in an incubator at 37 °C and 5% CO2 for 24 h. Subsequently, the medium was replaced with fresh medium containing 100 μg / mL iron and cultured for another 24 h. After that, the cells on the six-well plate were digested with trypsin and then lysed with RIPA lysis buffer containing protease inhibitors. The total protein content and ferritin content in the cells were measured using a BCA protein quantification kit and a Human FE ELISA Kit.

[0050] Cytotoxicity and cell uptake are shown Figure 4 as The left figure shows the cell viability. The results showed that after incubation for 24 h at iron concentrations from 0 to 500 μg / mL, the viability of HT-29 and Caco-2 cells remained above 80%.

[0051] The right figure shows the ferritin content (ng / mg) of Caco-2 cells during cell uptake. The results showed that the ferritin / total protein contents of Fe1-WPF and Fe2-WPF were 1.37 times and 1.17 times that of the ferrous sulfate control group, respectively. The iron uptake effect of Fe1-WPF was significantly higher than that of Fe2-WPF, indicating that Fe1-WPF has better bioavailability.

Claims

1. A preparation method of whey protein nanofibers loaded with nano iron oxides, characterized in that The method is as follows: (1) The whey protein isolate solution is placed at 0-4°C for hydration, the pH value is adjusted to acidic, heated and stirred for reaction, and then cooled to obtain a whey protein nanofiber solution; (2) Ferrous salts and ferric salts are added to the whey protein nanofiber solution, the pH value is adjusted to alkaline, and heated to 55-60°C for reaction to obtain whey protein nanofiber loaded with nano iron oxides.

2. The method according to claim 1, wherein In the step (1), the weight-volume concentration of the whey protein isolate solution is 2-3%, and after hydration, acid is added to adjust the pH value to 1.5-2.

3. The method according to claim 1, wherein In the step (1), the reaction is carried out at a temperature of 85-90°C for 5-8 hours.

4. The method according to claim 1, wherein In the step (2), the ferrous salts and ferric salts are sulfates, nitrates or chlorides.

5. The method according to claim 1, wherein In the step (2), the molar ratio of the ferrous salts to the ferric salts is 0.4-1.4:1; the total mass ratio of the ferrous salts and ferric salts to the mass of the solute in the whey protein nanofiber solution is 1:2-3.

6. The method according to claim 1, wherein In the step (2), ammonia water is added dropwise to adjust the pH value to 9-10, and the heating reaction time is 40-60 minutes.

7. The method according to claim 1, wherein The method includes the following steps: (1) Under stirring, dissolve whey protein isolate in deionized water to obtain a whey protein isolate solution with a weight-volume concentration of 2-3%, then place it at 0-4°C for 8-15 hours for hydration, then add acid to adjust the pH value to 1.5-2, heat and stir at 85-90°C for 5-8 hours, and cool to obtain a whey protein nanofiber solution; (2) Add ferrous salts and ferric salts to the whey protein nanofiber solution, add ammonia water dropwise under stirring to adjust the pH value to 9-10, then heat to 55-60°C for reaction for 40-60 minutes, and dialyze to remove the excess ammonia to obtain whey protein nanofiber loaded with nano iron oxides.

8. Whey protein nanofiber loaded with nano iron oxides prepared by the method according to any one of claims 1-7.

9. Application of the whey protein nanofiber loaded with nano iron oxides according to claim 8 as an iron supplement.

10. Application of the whey protein nanofiber loaded with nano iron oxides according to claim 8 in the preparation of iron supplement drugs or anti-anemia drugs.

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