Preparation method of phycocyanin polypeptide-ferrous chelate

Abstract

The invention relates to the technical field of peptide iron chelation processing, in particular to a preparation method of a phycocyanin polypeptide-ferrous chelate, which comprises the following steps: taking phycocyanin freeze-dried powder as a raw material, and carrying out enzymolysis by adopting protease combined with ultrasound to obtain phycocyanin polypeptide; dissolving with pure water to obtain phycocyanin polypeptide, adding ascorbic acid, and heating in a water bath; centrifuging a chelation reaction solution, taking supernate, adding ethanol, standing, centrifuging again, collecting precipitate, and performing vacuum freeze drying to obtain phycocyanin polypeptide-ferrous chelate powder; the phycocyanin polypeptide-ferrous chelate prepared by the invention has higher iron content, shows more excellent stability in the process of simulating gastrointestinal digestion in vitro compared with inorganic iron salt, further has triple functions of supplementing iron, resisting oxidation and supplementing essential amino acids, is prepared from food-grade raw materials, is safe in process and low in cost, and is suitable for industrial production. A low-cost and high-efficiency scheme is provided for IDA intervention, and the method can be widely applied to development of foods, medicines and health-care products.

Description

Technical Field

[0001] This invention relates to the field of peptide-iron chelation processing technology, specifically a method for preparing phycocyanin polypeptide-ferrous chelate. Background Technology

[0002] Iron is an essential trace element for the human body, a crucial component of hemoglobin and various metalloenzymes, and participates in a variety of physiological metabolic processes, including oxygen binding and transport, hematopoiesis and immune function maintenance, cell growth and differentiation, and gene regulation. Iron deficiency leads to iron deficiency anemia (IDA), one of the most common nutritional deficiency diseases worldwide.

[0003] To address the IDA problem, existing iron supplements on the market are mainly divided into two categories: inorganic iron salts and organic iron. However, both have significant drawbacks and fail to meet the needs for efficient and safe iron supplementation.

[0004] Inorganic iron salts (such as FeCl2 and FeSO4): Although they are lower in cost, they have three major problems. First, they have low absorption rates; second, they cause strong gastrointestinal irritation, leading to poor patient compliance; and third, they have poor stability and are easily oxidized into poorly absorbed Fe. 3+ .

[0005] Traditional organic iron (such as ferrous gluconate and ferrous fumarate): Although it reduces gastrointestinal irritation compared to inorganic iron salts, it still has limited bioavailability. Moreover, most products only have a single iron supplementation function and cannot simultaneously meet the body's multiple needs such as anti-oxidation and nutritional supplementation. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing phycocyanin polypeptide-ferrous chelate to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing phycocyanin polypeptide-ferrous chelate, comprising the following steps:

[0008] S1, Preparation of phycocyanin polypeptide:

[0009] S11. Raw material pretreatment: Take phycocyanin freeze-dried powder, dissolve it in ultrapure water at a substrate concentration of 25 g / L, and completely dissolve to obtain phycocyanin solution;

[0010] S12, Enzymatic hydrolysis-ultrasound synergistic treatment: Add protease to the phycocyanin solution to prepare an enzymatic hydrolysate, and simultaneously turn on ultrasound to assist enzymatic hydrolysis;

[0011] S13. Enzyme inactivation and centrifugation: After the enzymatic hydrolysis is completed, enzyme inactivation is performed, and the mixture is naturally cooled to room temperature. The supernatant is then collected by centrifugation.

[0012] S14. Freeze-drying: The supernatant was freeze-dried under vacuum to obtain powdered phycocyanin polypeptide, which was then sealed and stored at -20℃ for later use.

[0013] S2, chelation reaction:

[0014] S21. Preparation of polypeptide solution: Take the phycocyanin polypeptide prepared in step S14, dissolve it in ultrapure water, and prepare a polypeptide solution.

[0015] S22. Antioxidant Addition: Ascorbic acid is added to the peptide solution to prevent Fe... 2+ Oxidized to Fe 3+ ;

[0016] S23, pH adjustment and iron source addition: Adjust the pH of the mixture in step S22 to 4-9, and add anhydrous ferrous chloride (FeCl2, purity ≥99.5%) to the mixture at a peptide iron mass ratio of 6:1 to 1:3.

[0017] S24, Water bath chelation reaction: The mixture from step S23 is placed in a constant temperature water bath at 20-80℃ and heated for reaction;

[0018] S3, alcohol precipitation and drying:

[0019] S31. Initial centrifugation to remove impurities: After the chelation reaction in step S24 is completed, the reaction solution is cooled to room temperature, centrifuged to remove unreacted solid impurities, and the supernatant is collected.

[0020] S32, Alcohol precipitation: Slowly add 95% ethanol solution to the supernatant of step S31 at a volume ratio of 95% ethanol: supernatant = 4:1, and let stand at room temperature for 2 hours to allow the chelate to precipitate fully.

[0021] S33, Secondary centrifugation to collect the precipitate: Centrifuge the chelate from step S32, discard the supernatant, and collect the bottom precipitate;

[0022] S34. Freeze-drying: The precipitate from step S32 is freeze-dried under vacuum (under the same conditions as step S14) to obtain powdered phycocyanin polypeptide-ferrous chelate, which is then sealed and stored at -20℃ for later use.

[0023] Preferably, the protease in step S12 is one or more of neutral protease, alkaline protease, papain, or trypsin, used in combination.

[0024] During the ultrasound-assisted enzymatic hydrolysis process, the enzyme activity of the hydrolysate was 2400 U / mL, and the hydrolysis was carried out for 2 hours at pH 6–8 and temperature 37–60℃. The ultrasound-assisted parameters were 300W power and 20kHz frequency.

[0025] Preferably, the enzyme inactivation treatment in step S13 specifically involves heating the solution after enzymatic hydrolysis to 95°C and holding it for 10 minutes to inactivate the protease.

[0026] The centrifugation conditions are a rotation speed of 10,000 r / min and a centrifugation time of 10 to 15 min.

[0027] Preferably, in S14, the supernatant is frozen in an ultra-low temperature freezer at -80°C for 24 hours, and then transferred to a vacuum freeze dryer for 48 hours; the vacuum degree of the vacuum freeze dryer is ≤10Pa and the freezing temperature is ≤-50°C.

[0028] Preferably, the mass concentration of the polypeptide solution in step S21 is 0.2% to 4%; the ascorbic acid in step S22 accounts for 1% of the mass volume of the polypeptide solution; the pH adjustment in step S23 is specifically achieved by adjusting the pH of the mixture in step S22 with 1 mol / L HCl solution or 1 mol / L NaOH solution, and the inorganic iron salt is anhydrous ferrous chloride; the heating reaction time in step S24 is 20 to 140 min.

[0029] Preferably, the centrifugation conditions in step S31 are a rotation speed of 4000 r / min and a centrifugation time of 10 min;

[0030] In step S33, the centrifugation conditions are a rotation speed of 10,000 r / min and a centrifugation time of 15 min.

[0031] Compared with the prior art, the beneficial effects of the present invention are:

[0032] The phycocyanin peptide-ferrous chelate of this invention exhibits multiple advantages in addressing the high prevalence of iron deficiency anemia (IDA) and the shortcomings of existing iron supplements: its iron chelating activity reaches 189 mg / g, which is at a high level and superior to some reported peptide iron chelates. The peptide binds to Fe through amino acid residues. 2+ Coordination can effectively reduce Fe 2+ It binds with anti-nutritional factors such as phytic acid in food, thereby significantly improving bioavailability and solving the problem of low absorption of inorganic iron. In in vitro digestion experiments, the iron retention rate in the stomach was 47.58%, and in the small intestine it was 58.68%, significantly higher than that of common inorganic iron salts (such as FeCl2 and FeSO4), effectively preventing Fe from being absorbed. 2+ This process reduces gastrointestinal irritation and overcomes the shortcomings of traditional inorganic iron, such as poor stability and low bioavailability. Simultaneously, this chelate possesses both iron supplementation and antioxidant properties (DPPH free radical scavenging IC50). 50With its triple functions of providing 0.26 mg / mL of iron and supplementing essential amino acids, organic iron further expands its application value. Using food-grade phycocyanin as raw material, the process is controllable and safe, achieving high-value utilization of phycocyanin and providing a low-cost, high-efficiency new solution for nutritional intervention in IDA. Attached Figure Description

[0033] Figure 1 The chromatogram (A) and molecular weight distribution diagram (B) of the phycocyanin polypeptide prepared in this invention are shown.

[0034] Figure 2 Scanning electron micrographs of phycocyanin polypeptide (A) and phycocyanin polypeptide-ferrous chelate (B);

[0035] Figure 3 Infrared scan spectra of phycocyanin polypeptide (PC) and phycocyanin polypeptide-ferrous chelate (PCPI);

[0036] Figure 4 This is a schematic diagram illustrating the changes in iron content of the chelates prepared in Examples 2 to 6 under the influence of single-factor variables.

[0037] Figure 5 The DPPH radical scavenging rate (A) and ABTS radical scavenging rate (B) of phycocyanin polypeptide (PC) and phycocyanin polypeptide-ferrous chelate (PCPI) are respectively.

[0038] Figure 6 The iron retention rates after in vitro simulated digestion of phycocyanin polypeptide-ferrous chelate (PCPI), FeCl2, and FeSO4 were determined. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] In recent years, peptide iron complexes have become a research hotspot for novel iron supplements due to their high bioavailability, low side effects, and multifunctionality. The carboxyl, amino, and hydroxyl functional groups in peptide molecules can interact with Fe... 2+ Forming stable coordination bonds can prevent Fe 2+By binding to anti-nutritional factors in food, peptides can prevent their oxidation and achieve efficient iron delivery. Simultaneously, peptides can serve as a source of amino acids to supplement nutrition, and some peptides also possess antioxidant activities, achieving the dual value of "iron supplementation + function." However, current research on peptide-iron complexes largely focuses on animal-derived peptides (such as whey protein peptides and fish skin collagen peptides) or common plant-derived protein peptides (such as soybean protein peptides). These peptides generally suffer from insufficient chelating active sites or limited biological activity, resulting in low iron chelating activity and difficulty in simultaneously achieving multiple functions such as iron supplementation and high antioxidant activity.

[0041] Phycocyanin is a natural water-soluble pigment protein extracted from spirulina. It not only possesses excellent antioxidant, anti-inflammatory, and immunomodulatory activities, but also boasts a rich and balanced amino acid composition, with high levels of essential amino acids, particularly glutamic acid (Glu), aspartic acid (Asp), lysine (Lys), and histidine (His), which can form coordination bonds with iron ions, demonstrating excellent iron chelation potential. Existing research has confirmed that spirulina protein peptides can bind to ferrous ions to form complexes; however, studies using phycocyanin peptides as a carrier to prepare ferrous chelates, achieving a breakthrough in iron chelation activity while simultaneously providing multiple functions such as iron supplementation, antioxidant effects, and amino acid replenishment, have not yet been reported.

[0042] This invention aims to provide a method for preparing phycocyanin polypeptide-ferrous chelate. By optimizing the enzymatic hydrolysis process and chelation conditions, the iron chelation activity and stability of the product are improved, and a novel product with iron supplementation, antioxidant and nutritional supplementation functions is developed, providing a new approach for the high-value utilization of phycocyanin and the intervention of iron deficiency anemia.

[0043] Please see Figure 1-6 This invention provides a technical solution: a method for preparing phycocyanin polypeptide-ferrous chelate, comprising the following steps:

[0044] S1, Preparation of phycocyanin polypeptide:

[0045] S11. Raw material pretreatment: Take phycocyanin lyophilized powder and dissolve it in ultrapure water at a substrate concentration of 25 g / L to obtain phycocyanin solution;

[0046] S12. Enzymatic hydrolysis-ultrasound synergistic treatment: Add protease to the phycocyanin solution to prepare enzymatic hydrolysate, and simultaneously turn on ultrasound-assisted enzymatic hydrolysis. During the ultrasound-assisted enzymatic hydrolysis, the enzyme activity of the hydrolysate is 2400 U / mL, and the enzymatic hydrolysis is carried out for 2 hours under the conditions of pH 6–8 and temperature 37–60℃. The ultrasound-assisted parameters are power 300W and frequency 20kHz. The protease is one or more of neutral protease, alkaline protease, papain or trypsin.

[0047] S13. Enzyme inactivation and centrifugation: After the enzymatic hydrolysis is completed, the solution is heated to 95°C and kept at that temperature for 10 min to inactivate the protease. After naturally cooling to room temperature, it is centrifuged at 1000 r / min for 10 to 15 min and the supernatant is collected.

[0048] S14. Freeze-drying: The supernatant was placed in an ultra-low temperature freezer at -80℃ for 24 hours, and then transferred to a vacuum freeze dryer for 48 hours to obtain powdered phycocyanin peptides, which were then sealed and stored at -20℃ for later use; wherein the vacuum degree of the vacuum freeze dryer was ≤10Pa and the freezing temperature was ≤-50℃.

[0049] S2, chelation reaction:

[0050] S21. Preparation of polypeptide solution: Take the phycocyanin polypeptide prepared in step S14, dissolve it in ultrapure water, and prepare a polypeptide solution with a mass concentration of 0.2% to 4%.

[0051] S22. Antioxidant Addition: Add ascorbic acid to the peptide solution, wherein the ascorbic acid accounts for 1% of the peptide solution by mass and volume. Stir magnetically for 10 minutes until completely dissolved to prevent Fe... 2+ Oxidized to Fe 3+ ;

[0052] S23, pH adjustment and iron source addition: Adjust the pH of the mixture in step S22 to 4-9 using 1 mol / L HCl solution or 1 mol / L NaOH solution, and add anhydrous ferrous chloride (FeCl2, purity ≥99.5%) to the mixture at a peptide iron mass ratio of 6:1 to 1:3.

[0053] S24, Water bath chelation reaction: The mixture from step S23 is placed in a constant temperature water bath at 20-80℃ and heated for 20-140 min.

[0054] S3, alcohol precipitation and drying:

[0055] S31. Initial centrifugation to remove impurities: After the chelation reaction in step S24 is completed, the reaction solution is cooled to room temperature and centrifuged at 4000 r / min for 10 min to remove unreacted solid impurities and collect the supernatant.

[0056] S32, Alcohol precipitation: Slowly add 95% ethanol solution to the supernatant of step S31 at a volume ratio of 95% ethanol: supernatant = 4:1, and let stand at room temperature for 2 hours to allow the chelate to precipitate fully.

[0057] S33. Secondary centrifugation to collect the precipitate: The chelate from step S32 is centrifuged at 10,000 r / min for 15 min, the supernatant is discarded and the bottom precipitate is collected.

[0058] S34. Freeze-drying: The precipitate from step S32 is freeze-dried under vacuum (under the same conditions as step S14) to obtain powdered phycocyanin polypeptide-ferrous chelate, which is then sealed and stored at -20℃ for later use.

[0059] Example 1 (Selection of Protease)

[0060] Includes the following steps:

[0061] S1, Preparation of phycocyanin polypeptide:

[0062] S11. Raw material pretreatment: Take multiple groups of 5g phycocyanin freeze-dried powder, add 200mL of ultrapure water to each group to obtain multiple groups of phycocyanin solutions.

[0063] S12. Enzymatic hydrolysis-ultrasound synergistic treatment: Protease was added to the multiple groups of phycocyanin solutions to prepare enzymatic hydrolysate. At the same time, ultrasound-assisted enzymatic hydrolysis was turned on. During the ultrasound-assisted enzymatic hydrolysis, the enzyme activity of the enzymatic hydrolysate was 2400 U / mL. Enzymatic hydrolysis was carried out for 2 hours under the conditions of pH 6–8 and temperature 37–60℃. The ultrasound-assisted parameters were 300W power and 20kHz frequency.

[0064] S13. Enzyme inactivation and centrifugation: After the enzymatic hydrolysis is completed, the solution is heated to 95°C and kept at that temperature for 10 min to inactivate the protease. After naturally cooling to room temperature, it is centrifuged at 10000 r / min for 10 min and the supernatant is collected.

[0065] The proteases mentioned above include individual proteases such as neutral protease, alkaline protease, papain, and trypsin, as well as combinations of alkaline protease and neutral protease, combinations of alkaline protease and papain, combinations of papain and neutral protease, and combinations of alkaline protease, neutral protease, and papain. Enzymatic hydrolysis was carried out under the optimal reaction conditions shown in Table 1. The ferrous chelating ability of the different complex enzymatic hydrolysed phycocyanin polypeptide solutions was determined by the phenanthridine-ferrous complexation colorimetric method, and the results are shown in Table 1.

[0066] Table 1. Reaction conditions and ferrous binding capacity of different enzymes hydrolyzing peptides.

[0067]

[0068]

[0069] According to the results in Table 1, when alkaline protease, neutral protease and papain are used in combination, the ferrous chelating ability of the hydrolysate is the strongest. The degree of hydrolysis of the prepared phycocyanin peptide was determined by neutral formaldehyde titration. Under these conditions, the degree of hydrolysis of the peptide was 19.38% and the molecular weight (Mw) was 277410 Da.

[0070] Example 2 (Preparation of phycocyanin polypeptide-ferrous chelate using a complex protease of alkaline protease, neutral protease, and papain, with the iron source to polypeptide mass ratio during the chelation reaction serving as a control group)

[0071] Includes the following steps:

[0072] S1, Preparation of phycocyanin polypeptide:

[0073] S11. Raw material pretreatment: Take 5g of phycocyanin freeze-dried powder, add it to 200mL of ultrapure water, and stir magnetically for 30min until completely dissolved to obtain phycocyanin solution.

[0074] S12. Enzymatic hydrolysis-ultrasound synergistic treatment: Alkaline protease, neutral protease and papain are added to the phycocyanin solution in a mass ratio of 1:1:1 to form a complex protease and an enzymatic hydrolysate is prepared. At the same time, ultrasound-assisted enzymatic hydrolysis is turned on. During the ultrasound-assisted enzymatic hydrolysis, the enzyme activity of the enzymatic hydrolysate is 2400 U / mL, and the enzymatic hydrolysis is carried out at pH 7 and temperature 55℃ for 2 hours. The ultrasound-assisted parameters are power 300W and frequency 20kHz.

[0075] S13. Enzyme inactivation and centrifugation: After the enzymatic hydrolysis is completed, the solution is heated to 95°C and kept at that temperature for 10 min to inactivate the protease. After naturally cooling to room temperature, it is centrifuged at 10000 r / min for 10 min and the supernatant is collected.

[0076] S14. Freeze-drying: The supernatant was placed in an ultra-low temperature freezer at -80℃ for 24 hours, and then transferred to a vacuum freeze dryer for 48 hours to obtain powdered phycocyanin peptides, which were then sealed and stored at -20℃ for later use; wherein the vacuum degree of the vacuum freeze dryer was ≤10Pa and the freezing temperature was ≤-50℃.

[0077] The molecular weight and distribution of the phycocyanin peptides obtained above were determined by gel permeation chromatography (GPC). The specific method is as follows:

[0078] The sample was dissolved in distilled water and thoroughly mixed to prepare a 1 mg / mL solution. The prepared solution was filtered through a 0.45 μm microporous membrane, and 20 μL of the filtrate was injected into the GPC system using a microsyringe. The experimental conditions were: mobile phase of 0.1 mol / L NaNO3 aqueous solution, flow rate of 1 mL / min, and column temperature of 45 °C. During the determination of molecular weight and its distribution, polyethylene oxide (PEO) was used as the standard sample to establish a calibration curve. The ordinate of the calibration curve was converted to parameters related to molecular size to obtain a calibration curve suitable for the peptide sample. Based on the relationship between the calibration curve and the elution volume (VE) of the sample, the relative molecular weight and distribution of the phycocyanin peptide were calculated, and the results are shown in Table 2.

[0079] Table 2. Relative molecular weight distribution of phycocyanin peptides

[0080] RT(min) Mp(Da) Mw(Da) Mn(Da) Peak area percentage (%) polydispersity 8.052 236925 277410 191613 100％ 1.448

[0081] S2, chelation reaction:

[0082] S21. Preparation of polypeptide solutions: Take the phycocyanin polypeptide prepared in step S14, dissolve it in ultrapure water, prepare multiple polypeptide solutions with a mass concentration of 2%, and bring the volume to 50 mL.

[0083] S22. Antioxidant Addition: Add 0.5g of ascorbic acid to the above peptide solution to prevent Fe... 2+ Oxidized to Fe 3+ ;

[0084] S23, pH adjustment and iron source addition: Adjust the pH of the multiple mixed solutions in step S22 to 6 using 1 mol / L HCl solution or 1 mol / L NaOH solution, and add anhydrous ferrous chloride (FeCl2, purity ≥99.5%) to the multiple mixed solutions according to the iron to polypeptide mass ratios of 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2 and 1:3 respectively.

[0085] S24, Water bath chelation reaction: The multiple mixtures from step S23 are placed in a 50°C constant temperature water bath and heated for 40 minutes.

[0086] S3, alcohol precipitation and drying:

[0087] S31. Initial centrifugation to remove impurities: After the chelation reaction in step S24 is completed, the reaction solution is cooled to room temperature and centrifuged at 4000 r / min for 10 min to remove unreacted solid impurities and collect the supernatant.

[0088] S32, Alcohol precipitation: Slowly add 95% ethanol solution to the supernatant of step S31 at a volume ratio of 95% ethanol: supernatant = 4:1, and let stand at room temperature for 2 hours to allow the chelate to precipitate fully.

[0089] S33. Secondary centrifugation to collect the precipitate: The chelate from step S32 is centrifuged at 10,000 r / min for 15 min, the supernatant is discarded and the bottom precipitate is collected.

[0090] S34. Freeze-drying: The precipitate from step S32 is freeze-dried under vacuum (under the same conditions as step S14) to obtain powdered phycocyanin polypeptide-ferrous chelate, which is then sealed and stored at -20℃ for later use.

[0091] Example 3 (pH used as control group during chelation reaction)

[0092] The difference from Example 2 is:

[0093] S23. pH adjustment and iron source addition: The pH of the multiple mixed solutions in step S22 was adjusted to 4, 5, 6, 7, 8 and 9 respectively using 1 mol / L HCl solution or 1 mol / L NaOH solution. Anhydrous ferrous chloride (FeCl2, purity ≥99.5%) was added to each of the multiple mixed solutions at a mass ratio of iron to polypeptide of 1:1.

[0094] Example 4 (The mass fraction of the peptide solution during the chelation reaction serves as a control group)

[0095] The difference from Example 2 is:

[0096] S21. Preparation of polypeptide solutions: Take multiple groups of phycocyanin polypeptides prepared in step S14, dissolve them in ultrapure water, and prepare solutions with mass fractions of 0.2%, 0.4%, 0.6%, 0.8%, 1%, 2%, 3%, and 4%, respectively, and bring the volume up to 50 mL.

[0097] S22. Antioxidant Addition: Add 0.5g ascorbic acid to the above peptide solution and stir magnetically for 10 minutes until completely dissolved. This is to prevent Fe... 2+ Oxidized to Fe 3+ ;

[0098] S23. pH adjustment and iron source addition: Adjust the pH of the multiple mixed solutions in step S22 to 6 using 1 mol / L HCl solution or 1 mol / L NaOH solution, and add anhydrous ferrous chloride (FeCl2, purity ≥99.5%) to each of the multiple mixed solutions at a mass ratio of iron to peptide of 1:1.

[0099] Example 5 (Water bath temperature of chelation reaction as control group)

[0100] The difference from Example 2 is:

[0101] S23, pH adjustment and iron source addition: Adjust the pH of the multiple mixed solutions in step S22 to 6 using 1 mol / L HCl solution or 1 mol / L NaOH solution, and add anhydrous ferrous chloride (FeCl2, purity ≥99.5%) to the multiple mixed solutions at a mass ratio of iron to peptide of 1:1.

[0102] S24, Water bath chelation reaction: The multiple mixtures from step S23 are placed in water baths at constant temperatures of 30℃, 40℃, 50℃, 60℃, 70℃ and 80℃ for heating and reaction, with each heating reaction lasting 40 minutes.

[0103] Example 6 (Heating time in water bath for chelation reaction serves as control group)

[0104] The difference from Example 2 is:

[0105] S23, pH adjustment and iron source addition: Adjust the pH of the multiple mixtures in step S22 to 6 using 1 mol / L HCl solution or 1 mol / L NaOH solution, and add anhydrous ferrous chloride (FeCl2, purity ≥99.5%) to each of the multiple mixtures at a mass ratio of iron to peptide of 1:1, and stir magnetically for 5 min until FeCl2 is completely dissolved.

[0106] S24, Water bath chelation reaction: The multiple mixtures from step S23 are placed in a water bath at a constant temperature of 50°C and heated for 20 min, 40 min, 60 min, 80 min, 100 min, 120 min, and 140 min respectively, and stirred once every 20 min to ensure uniform reaction.

[0107] Example 7 (Preferred control group of Examples 2 to 6)

[0108] The iron content of the chelates prepared in Examples 2 to 6 was affected by single-factor variables in the experimental results, such as... Figure 4 As shown, based on Examples 2 to 6 of the implementation cases, four factors that have a greater impact on iron chelation activity were selected, and the optimal conditions for the experimental group were selected: iron peptide mass ratio 5:1, pH=8, peptide concentration 0.4%, temperature 50℃, and time 100min.

[0109] Based on the results of single-factor experiments in preparing chelates, factors with significant impact on iron chelation activity were selected. Following the Box-Behnken central composite principle in Design-Expert software, a four-factor, three-level response surface methodology (RSM) optimization experiment was designed. Using iron chelation activity as an indicator, the effects of the iron-to-titanium mass ratio (A), peptide concentration (B), pH (C), time (D), and their interactions on the chelation reaction were investigated to obtain the optimal preparation process for phycocyanin peptide ferrous chelates. Optimization using RSM yielded the optimal process parameters for the chelation reaction: an iron-to-peptide mass ratio of 5.2:1, pH 8.2, peptide concentration of 0.5%, reaction temperature of 50℃, and reaction time of 100 min. Under these conditions, the chelate prepared had the highest iron content, with an iron chelation activity reaching 189 mg / g.

[0110] The iron chelating activity of the phycocyanin polypeptide-ferrous chelates prepared in Examples 2 to 7 above was detected by o-phenanthroline spectrophotometry, as shown in Table 3.

[0111] Table 3 shows the optimal iron chelating activity of the phycocyanin polypeptide-ferrous chelates obtained in Examples 2 to 7.

[0112]

[0113]

[0114] As shown in Table 3, in Example 7, the chelation reaction process parameters were optimized using response surface methodology. The optimal conditions obtained were: iron peptide mass ratio of 5.2:1, pH 8.2, peptide concentration of 0.5%, reaction temperature of 50℃, and reaction time of 100 min. The phycocyanin peptide-ferrous chelate prepared under these conditions had the highest iron content, with an iron chelating activity of 189 mg / g. This result is significantly superior to the previously reported yak hide oligopeptide-ferrous chelate (iron chelating activity 42.72 mg / g) and sea cucumber-ferrous chelate (iron chelating activity 117.17 mg / g).

[0115] Example 8

[0116] This invention also provides structural characterization of phycocyanin peptides and phycocyanin peptide-ferrous chelates, the specific methods of which are as follows:

[0117] To verify the formation of phycocyanin peptide-iron chelate and to provide a basis for subsequent research on the structure-function relationship between peptide and iron, the obtained peptide-iron chelate was characterized by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR).

[0118] (1) Microstructure observation (SEM)

[0119] To compare the differences in microscopic morphology between phycocyanin peptides and phycocyanin peptide-ferrous chelates, this invention employs scanning electron microscopy (SEM) for analysis. Specifically, an appropriate amount of sample is adhered to a conductive adhesive, and gold is sputtered onto it using a sputtering method with a sputtering current of 5 mA and a sputtering time of 45 s. The treated sample is then placed in a scanning electron microscope and observed and images are acquired at 2000x magnification. Figure 2 As shown.

[0120] The observation results show that phycocyanin peptides mainly exhibit a blocky structure with a large particle size. Some blocky structures have small particles attached to their surface, and the overall surface is relatively smooth and regular. However, after chelation with ferrous ions, the resulting phycocyanin peptide-ferrous chelate mainly exists in the form of small particles. A small number of samples show a small blocky structure with even finer particles attached to its surface. The overall morphology is rougher and has irregular uneven features compared to the unchelated chelate.

[0121] (2) Fourier Transmission Infrared Spectroscopy (FTIR)

[0122] This invention employs the KBr pelleting method for Fourier transform infrared spectroscopy (FTIR) analysis of phycocyanin peptides and their ferrous chelates. The specific method is as follows: KBr is dried to constant weight in an infrared drying oven. 5 mg of phycocyanin peptide sample and 5 mg of phycocyanin peptide-ferrous chelate sample are accurately weighed and thoroughly ground with 500 mg of dried KBr powder. The ground mixture is then pressed into pellets using a pelleting machine, with KBr pellets without added samples used as a background control. The resulting pellets are scanned using an FTIR instrument at a wavenumber range of 4000–400 cm⁻¹. -1 .

[0123] The analysis results show that, Figure 3 As shown, compared with the unchelated phycocyanin peptide, the infrared spectrum of the chelate exhibits significant changes in several characteristic absorption regions. (3200–3500 cm⁻¹) -1 The changes in the intensity and shape of the broad peak in the region suggest that the hydrogen bonding environment of the N–H / O–H stretching vibration of the peptide chain underwent rearrangement or disruption after chelation; 1650 cm⁻¹ -1 (amideI, C=O stretching vibration) and 1540cm -1 The shift in the peak positions of amideII, N–H bending, and C–N stretching vibrations indicates that the carbonyl and amino groups in the peptide backbone are involved in the interaction with Fe. 2+ Coordination function. 1400–1600 cm -1 The asymmetric and symmetric stretching vibrations of the carboxylic acid group (–COO-) in the region suggest that the oxygen atom of the side chain carboxylic acid (derived from residues such as aspartic acid and glutamic acid) is the main coordination site. Furthermore, in the 400–600 cm⁻¹ region… -1The appearance of new absorption peaks in the region, which can be attributed to Fe–O and / or Fe–N stretching vibrations, further proves that Fe 2+ It forms stable coordination bonds with the carboxyl oxygen, carbonyl oxygen, and some nitrogen-containing groups in the peptide chain.

[0124] Example 9

[0125] This invention also provides analysis of the antioxidant properties of phycocyanin peptides and phycocyanin peptide-ferrous chelates, the specific methods of which are as follows:

[0126] The following method was used to determine the DPPH free radical scavenging ability of this invention: A 0.2 mmol / L DPPH solution was prepared using anhydrous ethanol. Phycocyanin polypeptide and phycocyanin polypeptide-ferrous chelate solutions with concentration gradients of 0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mg / mL were prepared respectively. 600 μL of the DPPH ethanol solution and 150 μL of the sample solution were added to a 2 mL centrifuge tube, mixed thoroughly, and reacted at room temperature in the dark for 30 min. After the reaction, the absorbance was measured at 517 nm using a UV-Vis spectrophotometer and recorded as A1; the absorbance was measured when anhydrous ethanol was mixed with the sample and recorded as A2; and the absorbance was measured when anhydrous ethanol was mixed with DPPH and recorded as A0. Vitamin C (Vc) was used as a positive control. The DPPH free radical scavenging rate was calculated according to the following formula:

[0127]

[0128] like Figure 5 As shown in Figure A, at the same concentration, the DPPH radical scavenging ability of the phycocyanin peptide chelate was significantly higher than that of the phycocyanin peptide itself. The IC50 values ​​for DPPH radical scavenging by the phycocyanin peptide and the phycocyanin peptide chelate were... 50 The values ​​were 3.14 mg / ml and 0.26 mg / ml, respectively.

[0129] The present invention uses the following method to determine the ABTS free radical scavenging ability: First, a 7 mmol / L ABTS stock solution is prepared for use. When using, the ABTS stock solution is diluted with 0.005 mol / L, pH 7.4 PBS buffer to a concentration of 0.7 ± 0.02 at room temperature and the absorbance is measured at 734 nm. This solution can then be used as the ABTS working solution.

[0130] Solutions of phycocyanin peptides and their ferrous chelates with concentration gradients of 0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mg / mL were prepared. For the assay, 600 μL of LABTS working solution was mixed with 60 μL of sample solution, precisely shaken for 30 seconds, and reacted at 37℃ in the dark for 6 min. The absorbance was then measured at 734 nm and recorded as A. 11 The absorbance was measured by mixing PBS with the sample and recorded as A.12 The absorbance of the ABTS working solution mixed with PBS was measured and recorded as A. 10 Vitamin C (Vc) was used as a positive control. The ABTS free radical scavenging rate was calculated using the following formula:

[0131]

[0132] like Figure 5 As shown in Figure B, at the same concentration, the ABTS radical scavenging ability of the phycocyanin peptide chelate is higher than that of the phycocyanin peptide itself. The IC50 values ​​for ABTS radical scavenging by the phycocyanin peptide and the phycocyanin peptide chelate are... 50 The values ​​were 0.21 mg / ml and 0.15 mg / ml, respectively.

[0133] In summary, phycocyanin peptide-ferrous chelate has good free radical scavenging ability and can be used as a potential antioxidant.

[0134] Example 10

[0135] This invention selects optimal preparation conditions for chelation rate to prepare chelates for in vitro simulated gastrointestinal digestion studies. The specific method is as follows:

[0136] To investigate the role of phycocyanin polypeptide carriers in the in vitro digestion of iron, this invention compares phycocyanin polypeptide-ferrous chelate with traditional inorganic iron salts (FeCl2, FeSO4).

[0137] (1) Salivary digestion: Prepare a 0.5% solution of phycocyanin polypeptide-ferrous chelate or FeCl2, FeSO4. Mix 10 mL of sample solution with 10 mL of diluted saliva and adjust the pH to 6.8. After preheating at 37 °C for 5 min, add salivary amylase (75 U / mL) and incubate on a shaker at 37 °C for 10 min (120 rpm).

[0138] (2) Gastric juice digestion: Take 10 mL of saliva-digested sample and mix it with 10 mL of diluted gastric juice. Quickly adjust the pH to 2.5, preheat at 37℃ for 5 min, add pepsin (2000 U / mL), and incubate on a shaker for 2 h (37℃, 120 rpm). Take an equal volume of sample reaction solution, add a small amount of 1 mol / L sodium bicarbonate to terminate the reaction, then add 4 times the volume of anhydrous ethanol and let it stand for 1 h to precipitate. Centrifuge at 8000 r / min for 20 min, take the supernatant and determine the free ferrous content, and calculate the iron retention rate accordingly.

[0139] (3) Small intestinal digestion: Take 10 mL of gastric juice to digest the sample and mix it with 10 mL of diluted small intestinal juice. Adjust the pH to 7.0, preheat at 37℃ for 5 min, add trypsin (100 U / mL) and lipase (2000 U / mL), and incubate on a shaker at 37℃ for 2 h (120 rpm). Take 5 mL of the sample reaction solution, inactivate the enzymes in a 95℃ water bath for 10 min, add 4 volumes of anhydrous ethanol, let stand for 1 h, centrifuge at 8000 r / min for 20 min, take the supernatant to determine the free ferrous iron content, and calculate the iron retention rate.

[0140] like Figure 6 As shown, the iron retention rate of the phycocyanin peptide-ferrous chelate was 47.58% after simulated gastric digestion, increasing to 58.68% after simulated small intestinal digestion. In the highly acidic environment of gastric juice, H... + Ions may react with Fe 2+ Competition for binding sites leads to the dissociation of some iron from the chelate, reducing iron retention. However, the increased pH in the small intestine favors the formation of peptide-mineral complexes, allowing some free iron ions to rebind to the peptides, enhancing chelate stability and significantly increasing iron retention. Overall, the iron retention rate of the phycocyanin peptide-ferrous chelate is significantly higher than that of FeCl2 and FeSO4, indicating that phycocyanin peptides have a protective effect during iron digestion.

[0141] Existing research has not reported the application of phycocyanin peptides in the development of iron chelating peptides. Therefore, this invention provides sufficient scientific basis for the development of novel iron supplements and the high-value-added utilization of phycocyanin resources. Experimental results show that the phycocyanin peptide-ferrous chelate has an IC50 value of [missing information - likely related to scavenging DPPH and ABTS free radicals]. 50 The antioxidant capacity of the chelated ferrous ion was significantly lower than that of the unchelated phycocyanin peptide, indicating that the antioxidant capacity of the peptide was significantly enhanced after chelation of ferrous ions. Furthermore, the chelate exhibited higher stability in in vitro simulated gastrointestinal digestion experiments, demonstrating a protective effect against ferrous ions and facilitating the effective delivery of ferrous ions to the required sites in the body.

[0142] The chelate prepared in this invention reacts with Fe through amino acid residues (containing carboxyl and amino groups) such as glutamic acid (Glu) and aspartic acid (Asp) of phycocyanin polypeptide. 2+ Forming stable coordination bonds to avoid Fe 2+ It combines with phytic acid and tannic acid in food to form insoluble precipitates (mechanistically eliminating the core reason for low inorganic iron absorption); and after optimization, the chelate's iron chelating activity reaches as high as 189 mg / g, significantly superior to existing peptide iron chelates (such as yak skin oligopeptide-ferrous chelate 42.72 mg / g and sea cucumber polypeptide-ferrous chelate 117.17 mg / g), and the polypeptide carrier can assist iron absorption through intestinal "peptide transporters". 2+ Absorption further enhances bioavailability.

[0143] The chelate prepared in this invention achieved an iron retention rate of 47.58% after simulated gastric digestion and 58.68% after simulated small intestinal digestion, while the small intestinal iron retention rates of inorganic iron salts FeCl2 and FeSO4 were only 15.56% and 25.18%, respectively—proving that phycocyanin peptides can "encapsulate" Fe during digestion. 2+ To prevent it from being oxidized by stomach acid into poorly absorbed Fe. 3+ (Solving the problem of poor stability of inorganic iron); because Fe 2+ Protected by peptide coordination, free Fe is avoided. 2+ It directly stimulates the gastrointestinal mucosa, reducing side effects such as nausea and diarrhea at the source.

[0144] The chelate prepared in this invention stably carries Fe 2+ It can directly supplement the human body with absorbable iron sources to improve IDA; the chelate scavenges DPPH free radicals. 50 =0.26 mg / mL, IC50 for scavenging ABTS free radicals 50 =0.15 mg / mL, significantly superior to unchelated phycocyanin peptides (DPPHIC) 50 =3.14 mg / mL, ABTSIC 50 =0.21mg / mL), which can simultaneously combat oxidative stress damage caused by iron deficiency in IDA patients; phycocyanin peptides themselves contain ≥40% essential amino acids (such as lysine and histidine), which can supplement the body with high-quality amino acids while supplementing iron, far exceeding the limitation of traditional organic iron "single iron supplementation". At the same time, the iron chelating activity and antioxidant capacity of phycocyanin peptide-ferrous chelate are higher than most existing peptide iron chelates, showing that it has the triple effects of iron supplementation, antioxidant and high-quality amino acid supplementation.

[0145] The chelate raw material prepared by this invention is food-grade phycocyanin (extracted from spirulina, naturally possessing antioxidant and anti-inflammatory activities, with no toxic risks), and the preparation process involves no chemical toxic reagents, and the product meets food and pharmaceutical grade standards.

[0146] This invention is the first to prepare iron chelates using phycocyanin peptides as carriers, transforming phycocyanin, which was originally used mainly in the pigment field, into a "functional iron supplement carrier," thereby increasing the added value of phycocyanin. Furthermore, the spirulina raw material is easy to cultivate on a large scale, providing a new iron supplementation solution with "low cost and high cost performance."

[0147] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0148] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a phycocyanin polypeptide-ferrous chelate, characterized in that: Includes the following steps: S1, Preparation of phycocyanin polypeptide: S11. Raw material pretreatment: Take phycocyanin lyophilized powder and dissolve it in ultrapure water at a substrate concentration of 25 g / L to obtain phycocyanin solution; S12, Enzymatic hydrolysis-ultrasound synergistic treatment: Add protease to the phycocyanin solution to prepare an enzymatic hydrolysate, and simultaneously turn on ultrasound to assist enzymatic hydrolysis; S13. Enzyme inactivation and centrifugation: After the enzymatic hydrolysis is completed, enzyme inactivation is performed, and the mixture is naturally cooled to room temperature. The supernatant is then collected by centrifugation. S14. Freeze-drying: The supernatant was freeze-dried under vacuum to obtain powdered phycocyanin polypeptide, which was then sealed and stored at -20℃ for later use. S2, chelation reaction: S21. Preparation of polypeptide solution: Take the phycocyanin polypeptide prepared in step S14, dissolve it in ultrapure water, and prepare a polypeptide solution. S22, Antioxidant Addition: Ascorbic acid is added to the peptide solution to prevent Fe2+. + Oxidized to Fe3 + ; S23, pH adjustment and iron source addition: Adjust the pH of the mixture in step S22 to 4-9, and add inorganic iron salt to the mixture at a peptide iron mass ratio of 6:1 to 1:

3. Stir magnetically for 5 minutes until the inorganic iron salt is completely dissolved. S24, Water bath chelation reaction: The mixture from step S23 is placed in a constant temperature water bath at 20-80℃ and heated for reaction; S3, alcohol precipitation and drying: S31. Initial centrifugation to remove impurities: After the chelation reaction in step S24 is completed, the reaction solution is cooled to room temperature, centrifuged to remove unreacted solid impurities, and the supernatant is collected. S32, Alcohol precipitation: Slowly add 95% ethanol solution to the supernatant of step S31 at a volume ratio of 95% ethanol: supernatant = 4:1, and let stand at room temperature for 2 hours to allow the chelate to precipitate fully. S33, Secondary centrifugation to collect the precipitate: Centrifuge the chelate from step S32, discard the supernatant, and collect the bottom precipitate; S34. Freeze-drying: The precipitate from step S32 is freeze-dried under vacuum to obtain phycocyanin polypeptide-ferrous chelate, which is then sealed and stored at -20℃ for later use.

2. The method for preparing a phycocyanin polypeptide-ferrous chelate according to claim 1, characterized in that: In step S12, the protease is one or more of the following: neutral protease, alkaline protease, papain, or trypsin, used in combination. During the ultrasound-assisted enzymatic hydrolysis process, the enzyme activity of the hydrolysate was 2400 U / mL, and the hydrolysis was carried out for 2 hours at pH 6–8 and temperature 37–60℃. The ultrasound-assisted parameters were 300W power and 20kHz frequency.

3. The method for preparing a phycocyanin polypeptide-ferrous chelate according to claim 1, characterized in that: The enzyme inactivation treatment in step S13 specifically involves heating the solution after enzymatic hydrolysis to 95°C and holding it for 10 minutes to inactivate the protease. The centrifugation conditions are a rotation speed of 10,000 r / min and a centrifugation time of 10 to 15 min.

4. The method for preparing a phycocyanin polypeptide-ferrous chelate according to claim 1, characterized in that: The vacuum freeze-drying process of the supernatant in S14 involves freezing the supernatant in an ultra-low temperature freezer at -80℃ for 24 hours, and then transferring it to a vacuum freeze dryer for 48 hours; the vacuum degree of the vacuum freeze dryer is ≤10Pa and the freezing temperature is ≤-50℃.

5. The method for preparing a phycocyanin polypeptide-ferrous chelate according to claim 1, characterized in that: In step S21, the mass concentration of the polypeptide solution is 0.2% to 4%; in step S22, ascorbic acid accounts for 1% of the mass volume of the polypeptide solution; in step S23, pH adjustment is specifically performed by using 1 mol / L HCl solution or 1 mol / L NaOH solution to adjust the pH of the mixture in step S22, and the inorganic iron salt is anhydrous ferrous chloride; in step S24, the heating reaction time is 20 to 140 min.

6. The method for preparing a phycocyanin polypeptide-ferrous chelate according to claim 1, characterized in that: In step S31, the centrifugation conditions are a rotation speed of 4000 r / min and a centrifugation time of 10 min. In step S33, the centrifugation conditions are a rotation speed of 10,000 r / min and a centrifugation time of 15 min.

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