Antarctic krill protein amyloid fibril-iron complex, preparation method therefor and use thereof

Abstract

The present invention relates to the technical field of food biology. Disclosed are an Antarctic krill protein amyloid fibril-iron complex, a preparation method therefor and a use thereof. In the present invention, Antarctic krill is used to extract Antarctic krill protein, and the Antarctic krill protein is induced by means of acid heat treatment to form Antarctic krill protein amyloid fibrils, which has valence protection and high iron-binding capacity. In addition, the complex has higher iron bioavailability than ferrous sulfate, and can relieve symptoms caused by iron deficiency anemia and alleviate gastric inflammation. The present invention provides a new idea for use of Antarctic krill protein, and the prepared Antarctic krill protein amyloid fibril-iron complex can be applied to iron-rich products and iron supplements.

Description

An Antarctic krill protein amyloid fiber-iron complex, its preparation method, and its applications. Technical Field

[0001] This invention relates to an Antarctic krill protein amyloid fiber-iron complex, its preparation method, and its application, belonging to the field of food biotechnology. Background Technology

[0002] Iron is an essential trace element for maintaining normal life activities in organisms. It is an important component of cytochromes, hemoglobin, and enzymes, playing a vital role in transporting oxygen and electrons. Iron deficiency is a major cause of anemia. Iron deficiency anemia is the most common type of anemia, causing fatigue, pale skin, and stunted growth. In severe cases, it can lead to difficulty breathing, angina, and other health problems, affecting people's quality of life and physical health.

[0003] Iron supplementation and food iron fortification are currently the dominant dietary strategies for addressing iron deficiency. The main source of iron in the human body is dietary iron from food; however, dietary iron is strongly interfered with during digestion and absorption by other food components such as phytates and polyphenols, leading to reduced iron bioavailability. Conventional iron fortifiers (such as ferrous sulfate) suffer from problems such as easy oxidation, poor sensory properties, and strong gastrointestinal irritation, severely limiting the application of iron-fortified foods and functional foods. In recent years, the use of functional components from food sources as carriers for food iron fortification has received widespread attention. Common examples include peptide-iron complexes and polysaccharide-iron complexes, which can improve the sensory properties of iron and increase its bioavailability. However, during digestion, due to H... + Competitive charging groups cause metal ions to dissociate, and the disrupted complexes lead to the release and dissolution of more iron. This released iron can irritate the gastric mucosa. During simulated digestion, iron is also easily oxidized or forms insoluble iron compounds under alkaline conditions, resulting in ineffective absorption in the small intestine. Therefore, exploring and developing iron-fortified foods that can achieve steady-state iron delivery while mitigating gastrointestinal irritation is a significant challenge.

[0004] Studies have shown that dietary amyloid fiber is safe and non-toxic. It is typically formed from the fibrillation of natural food proteins under conditions of low pH, low ionic strength, and high temperature. After protein fibrillation, the abundant active groups within the protein are exposed on the surface, allowing for the loading of nutritional functional substances through molecular interactions. Amyloid fiber exhibits greater resistance than natural proteins under extreme environments (such as acid, heat, and proteases). Furthermore, its specific steric hindrance gives it superior antioxidant properties, protecting the stable existence of nutritional functional substances. In recent years, dietary amyloid fibers have become excellent carriers for delivering nutrients. Examples include a method for encapsulating carotene using egg white lysozyme-amyloid fibers and improving carotene stability (Publication No.: CN112106988A), a method for preparing zeaxanthin dipalmitate high internal phase emulsions using protein-amyloid fiber-polysaccharide complexes (Publication No.: CN111467334A), and amyloid fiber-ferulic acid-chitosan dual-network hydrogels and their preparation methods and applications (Publication No.: CN115353646A). However, reports on amyloid fibers as mineral delivery carriers are scarce. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for preparing an Antarctic krill protein amyloid fiber-iron complex. The method involves extracting Antarctic krill protein and inducing its formation into Antarctic krill protein amyloid fibers through acid-heat treatment. This process protects the valence state of iron and can be applied to functional health products, fortified foods, and other fields. This invention uses an iron-deficiency anemia mouse model to evaluate the bioavailability of the Antarctic krill protein amyloid fiber-iron complex and its function in alleviating gastric inflammation.

[0006] The present invention provides a method for preparing an Antarctic krill protein amyloid fiber-iron complex, comprising the following steps:

[0007] (1) Extraction of Antarctic krill protein

[0008] Add deionized water to Antarctic krill meat, adjust the pH and stir evenly, centrifuge to collect the supernatant, adjust the pH of the supernatant to the isoelectric point of Antarctic krill protein and centrifuge to collect the precipitate, add water to the precipitate to adjust the pH, dialyze and freeze dry to obtain Antarctic krill protein.

[0009] (2) Formation of amyloid fibers in Antarctic krill

[0010] Antarctic krill protein solution prepared with deionized water was stirred evenly, pH was adjusted, heated, and freeze-dried to obtain Antarctic krill protein amyloid fibers.

[0011] (3) Formation of Antarctic krill protein amyloid fiber-iron complex

[0012] Antarctic krill protein amyloid fibers were dissolved in deionized water, ascorbic acid was added to adjust the pH, and iron salt was added to react. After the reaction was completed, the supernatant was collected by centrifugation, anhydrous ethanol was added, the precipitate was collected by centrifugation, washed and dried to obtain the Antarctic krill protein amyloid fiber-iron complex.

[0013] In one implementation method, step (1) of the preparation method of Antarctic krill protein specifically includes the following steps:

[0014] Add water to the Antarctic krill paste at a mass-to-volume ratio of 1 g: 5–10 mL. Adjust the pH to 11.0–12.5 and stir evenly for 2–3 hours. Centrifuge at 8000–10000 × g for 10–15 minutes at 4°C. Collect the supernatant and adjust its pH to the isoelectric point of the Antarctic krill protein with 6–8 mol / L hydrochloric acid solution. Centrifuge at 8000–10000 × g for 10–15 minutes at 4°C. Collect the precipitate and add 500–1000 mL of water to it. Stir evenly and adjust the pH to 7.0. Place the precipitate in a 3500 Da dialysis bag and dialyze for 24–48 hours. Freeze-dry to obtain the Antarctic krill protein.

[0015] In one implementation method, in step (2), the pH is adjusted to 2.0 to 3.0.

[0016] In one implementation method, step (2) involves heating at 70–90°C.

[0017] In one implementation method, in step (2), the mass concentration of the Antarctic krill protein solution is 1-5%.

[0018] In one implementation method, in step (3), the added iron salt is one of FeSO4·7H2O, FeCl2·4H2O or other inorganic iron salts.

[0019] In one implementation method, in step (3), the mass ratio of the added iron salt to the Antarctic krill protein amyloid fiber is 1:5 to 10:1, more preferably 1:2.

[0020] In one implementation method, in step (3), the reaction temperature is 20-60°C, more preferably 30°C; the reaction time is 20-60 min, more preferably 40 min.

[0021] In one implementation method, in step (3), the pH is adjusted to 2.0 to 7.0, more preferably 5.0.

[0022] In one implementation method, in step (3), the mass ratio of iron salt to ascorbic acid is in the range of 1:1 to 2.

[0023] The present invention provides an Antarctic krill protein amyloid fiber-iron complex prepared by the method described above.

[0024] This invention provides the application of the Antarctic krill protein amyloid fiber-iron complex described above in the preparation of iron-rich foods or iron supplements.

[0025] The beneficial effects of this invention are:

[0026] (1) This invention uses Antarctic krill protein as raw material to prepare Antarctic krill protein amyloid fiber, which expands the application range of protein and provides a reference for the high-value utilization of Antarctic krill.

[0027] (2) The Antarctic krill starch-like fiber prepared by the present invention has a protective effect on the valence state of iron, and the resulting Antarctic krill protein starch-like fiber-iron complex has a high iron content, with the highest iron content reaching about 60%.

[0028] (3) The Antarctic krill amyloid fiber-iron complex prepared by the present invention can improve the bioavailability of iron, alleviate iron deficiency anemia and gastric inflammation, and provide a basis for the application of Antarctic krill amyloid fiber-iron complex.

[0029] (4) The preparation method of the present invention is simple and quick, and the prepared Antarctic krill starch-iron complex can be used in the fields of iron supplements, fortified foods and other fields. Attached Figure Description

[0030] Figure 1 is a thiosulfate T fluorescence image of Antarctic krill starch-like fibers in Example 1;

[0031] Figure 2 shows the microstructure of the starch-like fibers of Antarctic krill in Example 1;

[0032] Figure 3 shows the iron reduction capacity of Antarctic krill starch-like fibers in Example 1;

[0033] Figure 4 is the infrared spectrum of the Antarctic krill starch-fiber-iron complex in Example 1;

[0034] Figure 5 is the fluorescence spectrum of the Antarctic krill starch-iron complex in Example 1;

[0035] Figure 6 is the energy spectrum of the Antarctic krill starch-iron complex in Example 1;

[0036] Figure 7 shows the effect of Antarctic krill amyloid fiber-iron complex on body weight in mice with iron deficiency anemia in Example 1.

[0037] Figure 8 shows the effect of Antarctic krill amyloid fiber-iron complex on the iron content in the spleen of mice with iron deficiency anemia in Example 1.

[0038] Figure 9 shows the effect of Antarctic krill amyloid fiber-iron complex on the liver of mice with iron deficiency anemia in Example 1.

[0039] Figure 10 shows the effect of Antarctic krill amyloid fiber-iron complex on the spleen of mice with iron deficiency anemia in Example 1.

[0040] Figure 11 shows the effect of Antarctic krill amyloid fiber-iron complex on the expression of iron homeostasis genes in iron-deficiency anemia mice in Example 1.

[0041] Figure 12 shows the effect of Antarctic krill amyloid fiber-iron complex on the expression of gastric inflammatory genes in iron-deficiency anemic mice in Example 1. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, but the implementation and protection scope of the present invention are not limited thereto. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used that do not specify the manufacturer are all commercially available conventional products. All raw materials used in the embodiments are food-grade.

[0043] Example 1: A method for preparing an Antarctic krill protein amyloid fiber-iron complex

[0044] (1) Extraction of Antarctic krill protein (AKP)

[0045] Water was added to the Antarctic krill meat paste at a mass-to-volume ratio of 1 g: 10 mL. The pH was adjusted to 12.5, and the mixture was stirred evenly for 2 hours. The mixture was then centrifuged at 10,000 × g for 10 minutes at 4°C. The supernatant was collected, and the pH of the supernatant was adjusted to the isoelectric point of the Antarctic krill protein using 6 mol / L hydrochloric acid solution. The mixture was then centrifuged at 10,000 × g for 10 minutes at 4°C. The precipitate was collected, and 500 mL of water was added to the precipitate. The mixture was stirred evenly, and the pH was adjusted to 7.0. The mixture was then placed in a 3500 Da dialysis bag and dialyzed for 48 hours. The resulting Antarctic krill protein was obtained by freeze-drying.

[0046] (2) Preparation of Antarctic krill protein amyloid fibers (AKAF)

[0047] An Antarctic krill protein solution with a mass fraction of 1% was prepared by deionized water, stirred uniformly at 4°C for 6 hours, the pH was adjusted to 2.0, heated at 90°C for 15 minutes, and then freeze-dried to obtain Antarctic krill protein amyloid fibers.

[0048] (3) A method for preparing an Antarctic krill protein amyloid fiber-iron complex (AKAF-Fe).

[0049] Antarctic krill protein amyloid fibers were dissolved in deionized water to prepare an Antarctic krill protein amyloid fiber solution (1%, w / v). Ascorbic acid was added to the solution to prevent Fe from forming in the system. 2+ Oxidation was performed, pH was adjusted to 5, and FeSO4·7H2O was added and mixed. The mass ratio of FeSO4·7H2O to ascorbic acid was 1:2, and the mass ratio of FeSO4·7H2O to Antarctic krill protein amyloid fibers was 1:2. The system was reacted at 30℃ for 40 min, the supernatant was collected by centrifugation, and 6 volumes of anhydrous ethanol were added. The mixture was allowed to stand to allow complete precipitation. The ethanol was removed by centrifugation to obtain the AKAF-Fe complex, which was washed with ethanol. This operation was repeated 4 times to remove excess iron ions. The remaining ethanol was evaporated by nitrogen blowing to obtain AKAF-Fe powder with an iron content of 60.66%.

[0050] Example 2

[0051] The preparation method is the same as that in Example 1, except that the pH adjusted in step (3) is replaced with 2, 3, 4, 6 and 7 respectively.

[0052] Experimental results: The iron content of Antarctic krill protein amyloid fiber-iron complexes prepared under pH conditions of 2, 3, 4, 6, and 7 were 4.96%, 9.57%, 55.67%, 46.64%, and 39.68%, respectively, which were lower than the iron content of Antarctic krill protein amyloid fiber-iron complexes prepared under pH condition of 5.

[0053] Example 3

[0054] The preparation method of Example 1 is the same, except that the reaction time in step (3) is replaced with 20 min, 30 min, 50 min and 60 min respectively.

[0055] Experimental results: The iron content of the Antarctic krill protein amyloid fiber-iron complex prepared under reaction times of 20 min, 30 min, 50 min and 60 min were 47.58%, 56.71%, 40.62% and 42.03% respectively, which were lower than the iron content of the Antarctic krill protein amyloid fiber-iron complex prepared under the reaction time of 40 min.

[0056] Example 4

[0057] The preparation method of Example 1 is the same, except that the reaction temperature in step (3) is replaced with 20℃, 40℃, 50℃ and 60℃ respectively.

[0058] Experimental results: The iron content of the Antarctic krill protein amyloid fiber-iron complex prepared at reaction temperatures of 20℃, 40℃, 50℃ and 60℃ was 52.57%, 52.38%, 51.63% and 50.69%, respectively, which was lower than the iron content of the Antarctic krill protein amyloid fiber-iron complex prepared at a reaction temperature of 30℃.

[0059] Example 5

[0060] The preparation method is the same as in Example 1, except that in step (3), the mass ratio of Antarctic krill protein amyloid fiber to FeSO4·7H2O is replaced with 1:10, 1:5, 1:2, and 5:1, respectively.

[0061] Experimental results: The iron content of the Antarctic krill protein amyloid fiber-iron complex prepared under the conditions of Antarctic krill protein amyloid fiber to FeSO4·7H2O mass ratios of 1:10, 1:5, 1:2, and 5:1 were 37.23%, 43.16%, 47.11%, and 57.74%, respectively, which were lower than the iron content of the Antarctic krill protein amyloid fiber-iron complex prepared under the condition of a mass ratio of 2:1.

[0062] Comparative Example 1

[0063] The preparation method is the same as in Example 1, except that Antarctic krill protein is replaced with soy protein isolate.

[0064] Comparative Example 2

[0065] The preparation method is the same as in Example 1, except that Antarctic krill protein is replaced with pea protein.

[0066] The iron contents of the complexes obtained in Comparative Examples 1 and 2 were 42.48% and 55.77%, respectively, which were lower than the iron contents of the Antarctic krill protein amyloid fiber-iron complex prepared in Example 1.

[0067] The following characterization and performance tests were performed on AKAF and AKAF-Fe prepared in Example 1.

[0068] 1. Thiamine T fluorescence image of Antarctic krill amyloid fibrils (AKAF)

[0069] 8 mg of thioflavin T (ThT) powder was dissolved in 10 mL of phosphate buffer solution (PB, 10 mM, pH 7.0, 150 mM NaCl). Undissolved ThT was removed using a 0.22 μm filter and stored at 4 °C protected from light. On the day of analysis, the ThT stock solution was diluted 50-fold with PB to prepare the ThT working solution. The sample solution (0.1 mg / mL) and the ThT working solution were mixed at a volume ratio of 1:100 and equilibrated for 2 min in the dark. The excitation wavelength was 440 nm using a fluorescence spectrophotometer (1500 nm / min), and the ThT fluorescence intensity of the working solution was used as the fluorescence background value.

[0070] Experimental Results: As shown in Figure 1, the ThT fluorescence intensity of AKAF initially increased and then decreased with prolonged heating time. The increase in fluorescence intensity indicates that AKAF formed an ordered β-sheet structure after heating for 15 min. The heating time to reach maximum fluorescence intensity at 90℃ was four times that at 70℃, indicating that high temperature can accelerate fiber formation. This is consistent with existing results: due to the characteristics of protein denaturation at high temperatures, higher temperatures accelerate the rearrangement of protein structural units, increase the nucleation rate of fibers, and thus promote fiber formation. However, after heating for 30 min, the fluorescence intensity of ThT decreased. This phenomenon may be attributed to two reasons: first, excessive hydrolysis leads to the destruction of the ordered fiber structure; second, fiber aggregation shields the ThT binding sites.

[0071] 2. Microscopic morphology of Antarctic krill protein amyloid fibers

[0072] A 10 μL sample was dropped onto the sample stage and rapidly frozen with liquid nitrogen. After sublimation (-90 °C, 30 min), the sample was cut to form a cross-section, and gold was sputtered onto the surface. The microstructure of the sample was observed at 10 kV.

[0073] Experimental results: Under cold field scanning electron microscopy, unheated AKP was observed to contain globular proteins and protein fragments of varying sizes. After 15 min, AKP exhibited a slender, straight fibrous shape (Figure 2).

[0074] 3. The reducing power of iron by Antarctic krill protein amyloid fibers

[0075] 10 μL of sample (2 mg / mL and 20 mg / mL) was mixed with 30 μL of 0.1 M FeCl3·6H2O, and then 1.8 mL of 5 mM o-phenanthroline was added. The mixture was incubated at 37 °C for 8 h, and measurements were taken at 512 nm every 30 min. Fe-free and fiber-free samples were used as controls to eliminate the influence of the reaction system itself.

[0076] Experimental Results: The reduction effect of AKAF at different concentrations on iron was investigated, as shown in Figures 3A (2 mg / mL) and 3B (20 mg / mL). We observed that at both concentrations, the iron reduction capacity of AKAF increased with increasing reaction time. The results indicate that fibrils possess the ability to reduce iron, and this reduction effect is concentration-dependent. Therefore, AKAF can be considered an ideal candidate for iron fortification because it can maintain iron in a more bioavailable iron(II) state.

[0077] 4. Determination of iron content in Antarctic krill protein-amyloid fiber-iron complex

[0078] The iron content of AKAF-Fe was determined using the o-phenanthroline colorimetric method. A 1 mg / mL iron standard solution was serially diluted to solutions of 0, 0.02, 0.04, 0.06, 0.08, and 0.1 mg / mL. 1 mL of 0.2 mol / L acetate-sodium acetate buffer (pH 5.0) and 200 μL of 20 mg / mL ascorbic acid were added sequentially to each solution. After thorough mixing, 400 μL of 5 mg / mL o-phenanthroline solution was added to prepare the working colorimetric solution. The reaction was allowed to stand for 15 min. Ultrapure water was used as a blank control. The absorbance of the reaction solution was measured at 510 nm. Based on the Fe... 2+ A standard curve was plotted using concentration and absorbance values. To dissociate iron ions, the AKAF-Fe powder was acidified with a small amount of 6M HCl and then diluted to volume with water. The working solution was added sequentially as described above, and the absorbance of the sample was measured at 510 nm. The iron content in the solution was then determined using the standard curve. The iron content in the sample was calculated using the formula:

[0079] Iron content = (Iron content in solution * Solution volume) / Total sample mass * 100%

[0080] 5. Infrared spectrum of Antarctic krill protein amyloid fiber-iron complex (AKAF-Fe)

[0081] The sample was mixed with potassium bromide, ground, and extruded into sheets. The sample was then scanned using a Fourier transform infrared spectroscopy (FTIR) instrument. The scans were performed in the range of 400–4000 cm⁻¹. -1 Range scanning, instrument resolution is 4cm -1 Infrared spectra were obtained.

[0082] Experimental Results: Figure 4 shows that the -NH and -OH groups of AKAF form new coordinate bonds with iron, and the C=O group also participates in the binding interaction. This binding interaction is attributed to the binding of iron ions by the unbonded free electrons of the carbonyl oxygen. Furthermore, the red shift of the characteristic peak of the –COO- group in AKAF may be due to the formation of –COO-Fe. Therefore, the iron binding site may be related to the C-terminal carboxyl group, glutamic acid, and aspartic acid residues. Meanwhile, at 1131 cm⁻¹... -1A strong band was also found in AKAF-Fe, which is likely due to the formation of C–O–Fe bonding sites. AKAF-Fe exhibits a band at 618 cm⁻¹. -1 The characteristic peaks are attributed to the C–N bond stretching vibration, which is due to the bonding of the N atom in the imidazole group with iron. Therefore, the bonding of AKAF with iron mainly occurs on the carboxyl, amino, hydroxyl, and imidazole groups.

[0083] 6. Fluorescence spectrum of Antarctic krill protein amyloid fibrils-iron complex (AKAF-Fe)

[0084] The spectral information of the sample was determined using a fluorescence spectrophotometer under the conditions of excitation wavelength of 280 nm, scanning range of 290–450 nm, and scanning speed of 1500 nm / min.

[0085] Experimental results: As shown in Figure 5, the introduction of iron significantly reduced the fluorescence intensity of AKAF itself, quenched the intrinsic fluorescence of the protein, and was accompanied by a shift in the maximum absorption peak. This indicates that protein folding occurred during binding, and there is a strong interaction and energy transfer between AKAF and iron.

[0086] 7. Energy dispersive spectroscopy of Antarctic krill protein amyloid fibrils-iron complex (AKAF-Fe)

[0087] The sample powder was uniformly coated onto an adhesive tape, and a gold sputtering treatment was performed at a 15mA point for 90 seconds. Scanning was then performed using an energy-dispersive X-ray spectrometer at an accelerating voltage of 5kV.

[0088] Experimental Results: The energy dispersive spectroscopy (EDS) spectra and relative elemental contents of AKAF and AKAF-Fe are shown in Figures 6A and 6B. AKAF clearly shows the presence of C, N, and O, the three basic protein building blocks, with contents of 56.80%, 24.07%, and 17.49%, respectively. AKAF-Fe contains 19.79% iron, indicating that AKAF can bind to iron.

[0089] 8. Establishment of a mouse model of iron deficiency anemia

[0090] Forty SPF-grade, three-week-old female C57BL / 6 mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd. and housed in an SPF-grade animal laboratory. During the experiment, the mice had free access to food and water. After one week of acclimatization, 10 mice were randomly selected and fed a normal diet (45 ppm Fe) until the end of the experiment; this group was designated as the normal control group (NC). The experimental group mice were fed a low-iron diet (12 ppm Fe) to establish an iron deficiency anemia mouse model. During the eight-week period, blood was collected from the orbital region of the mice and measured using a hemoglobin (Hb) kit (Nanjing Jiancheng). An Hb level less than 100 g / L is generally considered to indicate iron deficiency anemia. After the iron deficiency model was established, the experimental group was randomly divided into three subgroups based on hemoglobin levels and mouse weight, and continued to be fed a low-iron diet for three weeks. During this period, the normal control group (NC) and the iron deficiency model group (MD) were given deionized water daily. The remaining two groups received additional iron intake via daily gavage with FeSO4 solution (2.3 ppm Fe) and AKAF-Fe solution (2.3 ppm Fe), respectively, designated as the FE group and the AF group. Mice body weight was recorded weekly for 3 weeks of iron supplementation. Eighteen hours after the last gavage, the mice were euthanized, and their livers, spleens, and stomachs were collected for subsequent experiments.

[0091] Experimental Results: The changes in mouse body weight are shown in Figure 7. After the iron-deficient diet (week 0), there was no significant difference in body weight between the iron-deficient diet group and the normal control group, but the body weight was significantly reduced. Therefore, IDA affects the growth and development of mice. Then, IDA mice were supplemented with iron by ingesting FeSO4 and AKAF-Fe for 3 consecutive weeks. We found that the body weight of the AF group was significantly different from that of the MD and FE groups, indicating that AKAF-Fe can improve the problem of slow growth and development in mice and is more effective than free iron alone.

[0092] 9. Effect of Antarctic krill protein amyloid fiber-iron complex (AKAF-Fe) on spleen iron content in iron-deficiency anemic mice.

[0093] A 0.1g spleen was weighed and digested using a wet digestion method to form a working solution. The iron content in the tissue was then determined by atomic absorption spectrophotometry.

[0094] Experimental results: IDA caused a decrease in iron content in the spleen of mice (Figure 8). Ingestion of FeSO4 and AKAF-Fe alleviated spleen hypoplasia and iron storage capacity, restoring them to normal levels. Compared with mice ingesting FeSO4, AKAF-Fe showed a better effect in intervening in iron storage capacity.

[0095] 10. Effects of Antarctic krill protein amyloid fiber-iron complex (AKAF-Fe) on organs of mice with iron deficiency anemia.

[0096] The liver and spleen were fixed with 4% paraformaldehyde solution. After embedding and sectioning, the tissue sections were stained with hematoxylin-eosin and the pathological changes of the tissues were observed under an optical microscope.

[0097] Experimental Results: As shown in Figure 9, normal mouse hepatocytes were regularly arranged and morphologically intact. Compared with the NC group, we observed indistinct hepatocyte boundaries, disordered sinusoidal dilation, and mononuclear cell infiltration and inflammatory accumulation (black arrows) in the MD group. This indicates that IDA can damage the liver and induce an inflammatory response. Obvious lipid droplet vacuoles and inflammatory accumulation (black arrows) were observed in the FE group, indicating that FeSO4 intake induces steatosis and inflammatory response. However, there was no significant difference between the AKAF-Fe intake group and the normal group. This suggests that AKAF-Fe has a better intervention effect on liver damage.

[0098] Figure 10 shows that the main effect of IDA on the spleen is the formation of well-formed macrophages and inflammatory cells (red circles) accompanied by organ damage. Compared with the NC group, no obvious lesions were observed in the FE group and the AKAF-FE group. However, well-formed macrophages and inflammatory cells (red circles) were also observed in the FE group, indicating that FeSO4 is toxic.

[0099] 11. Real-time quantitative PCR experiment

[0100] Total RNA was extracted using Trozol reagent (Sangon, Shanghai, China). gDNA removal and cDNA synthesis were performed using the Evo-MLV reverse transcription kit (Accurate Biology, Hunan, China) on a T100 Thermal Cycler (BIO-RAD) according to the manufacturer's instructions. Subsequently, real-time quantitative PCR was performed using the SYBR Green premix pro Taq HS qPCR kit (Accurate Biology, Hunan, China) via the SYBR Green I chimeric fluorescence assay. Two... -ΔΔCt The relative gene expression levels were calculated using a method. The primer sequences used are shown in the table below.

[0101] Table 1 Primer sequences used

[0102] (1) Effects of Antarctic krill amyloid fiber-iron complex on iron homeostasis gene expression in mice

[0103] Experimental Results: DMT1 is the main absorption pathway for non-heme iron in the intestine. IDA induces an increase in DMT1 mRNA levels. As shown in Figure 11A, mice ingesting AKAF-Fe (AF group) showed a significant difference in DMT1 mRNA expression compared to the MD group, indicating that AKAF-Fe can positively affect DMT1 expression. FPN1 is a known non-heme iron export protein in mammals, located in the basolateral intestinal membrane. Figure 11B shows that compared to the MD group, the FPN1 mRNA expression levels in mice in the FE and AF groups were significantly reduced and then returned to normal levels. The significant difference in FPN1 mRNA expression between the AF and FE groups indicates that AKAF-Fe intake has a better restorative effect on FPN1.

[0104] (2) Effects of Antarctic krill amyloid fiber-iron complex on gene expression of inflammatory factors in the stomach of mice;

[0105] Experimental Results: Inflammatory factors in the stomach were investigated, including pro-inflammatory factors IL-1β and IL-6, and anti-inflammatory factor IL-10. As shown in Figure 12, compared with the normal control group, the mRNA expression levels of IL-1β and IL-6 were significantly increased, while the mRNA expression level of IL-10 was significantly decreased in the MD group, indicating that IDA induced an inflammatory response in the stomach. Conversely, iron supplementation effectively reduced IL-1β and IL-6 levels and increased IL-10 levels. Specifically, compared with the FE group, AKAF-Fe intake had a more positive regulatory effect on IL-1β, IL-6, and IL-10, indicating that AKAF-Fe can better alleviate the inflammatory response in the stomach than FeSO4. Based on this, AKAF, as a carrier, can effectively protect the stomach from iron stimulation.

[0106] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for preparing an Antarctic krill protein amyloid fiber-iron complex to alleviate iron deficiency anemia and gastric inflammation, characterized in that, Includes the following steps: (1) Extraction of Antarctic krill protein Deionized water was added to Antarctic krill meat, the pH was adjusted and stirred evenly, the supernatant was collected by centrifugation and the pH was adjusted to the isoelectric point of Antarctic krill protein. The precipitate was collected by centrifugation, water was added to the precipitate to adjust the pH, and after dialysis, it was freeze-dried to obtain Antarctic krill protein. (2) Formation of amyloid fibers in Antarctic krill Antarctic krill protein solution was prepared with deionized water, and after uniform stirring, the pH was adjusted, heated, and freeze-dried to obtain Antarctic krill protein amyloid fibers. (3) Preparation of Antarctic krill protein amyloid fiber-iron complex Antarctic krill protein amyloid fibers were dissolved in deionized water, ascorbic acid was added to adjust the pH, and iron salt was added to react. After the reaction was completed, the supernatant was collected by centrifugation, anhydrous ethanol was added, the precipitate was collected by centrifugation, washed and dried to obtain the Antarctic krill protein amyloid fibers-iron complex. The mass ratio of added iron salt to Antarctic krill protein amyloid fibers was 1:5 to 10:1; the reaction temperature was 20 to 60℃; and the reaction time was 20 to 60 min.

2. The preparation method according to claim 1, characterized in that, In step (1), the preparation method of Antarctic krill protein specifically includes the following steps: Add water to the Antarctic krill paste at a mass-to-volume ratio of 1 g: 5–10 mL. Adjust the pH to 11.0–12.5 and stir evenly for 2–3 hours. Centrifuge at 8000–10000 × g for 10–15 minutes at 4°C. Collect the supernatant and adjust its pH to the isoelectric point of the Antarctic krill protein with 6–8 mol / L hydrochloric acid solution. Centrifuge at 8000–10000 × g for 10–15 minutes at 4°C. Collect the precipitate and add 500–1000 mL of water to it. Stir evenly and adjust the pH to 7.

0. Place the precipitate in a 3500 Da dialysis bag and dialyze for 24–48 hours. Freeze-dry to obtain the Antarctic krill protein.

3. The preparation method according to claim 1, characterized in that, In step (2), the pH is adjusted to 2.0 to 3.0; the heating temperature is 70 to 90°C.

4. The preparation method according to claim 1, characterized in that, In step (2), the mass concentration of the Antarctic krill protein solution is 1-5%.

5. The preparation method according to claim 1, characterized in that, In step (3), the mass ratio of the added iron salt to the Antarctic krill protein amyloid fiber is 1:

2.

6. The preparation method according to claim 1, characterized in that, In step (3), the reaction temperature is 30℃ and the reaction time is 40min.

7. The preparation method according to claim 1, characterized in that, In step (3), the pH is adjusted to 2.0 to 7.

0.

8. The preparation method according to claim 1, characterized in that, In step (3), the mass ratio of iron salt to ascorbic acid is 1:1 to 2.

9. The Antarctic krill protein amyloid fiber-iron complex prepared by any one of the methods described in claims 1 to 8.

10. The use of the Antarctic krill protein amyloid fiber-iron complex according to claim 9 in the preparation of iron-rich foods or iron supplements.

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