Sheep blood phosphopeptide chelated iron prepared by one-pot method as well as preparation method and application of sheep blood phosphopeptide chelated iron

Abstract

The invention discloses sheep blood phosphopeptide chelated iron prepared by a one-pot method, a preparation method and application, and relates to the technical field of preparation methods of peptide chelated iron, the method comprises the following steps: dissolving sheep blood powder in water, and preheating to 50-55 DEG C to obtain a sheep blood powder solution; papain is added into the sheep blood powder solution for enzymolysis, a sheep blood peptide solution is obtained, sodium pyrophosphate is added into the sheep blood peptide solution for phosphorylation modification, and a sheep blood phosphopeptide reaction solution is obtained; adjusting the pH value of the sheep blood phosphopeptide reaction liquid to 3.0-4.0 to dissociate heme groups to obtain dissociation liquid; and centrifuging the dissociation liquid, and recovering the supernatant to obtain the sheep blood phosphopeptide chelated iron. In the preparation process, endogenous iron ions contained in the heme are utilized, so that the complex preparation process of the current blood peptide iron chelate is simplified.

Description

Technical Field

[0001] The invention relates to the technical field of preparation methods of peptide chelated iron, and more specifically to sheep blood phosphopeptide chelated iron prepared by a one-pot method, a preparation method and application thereof. Background Art

[0002] Iron is an essential trace element in living organisms, affecting the synthesis of hemoglobin, immunity and cardiopulmonary function. Iron deficiency is a worldwide nutritional problem and the most common cause of anemia. The main causes of iron deficiency anemia are insufficient dietary iron intake, impaired iron absorption or excessive iron loss. Currently, many finely processed foods lack trace elements, and diet alone cannot meet the body's iron needs. On the other hand, the absorption of iron in the human body has problems such as low efficiency and poor stability. Therefore, food-derived iron supplements have always been the focus of the food industry.

[0003] Among the various strategies for controlling iron deficiency, food fortification is an effective measure to increase the content of nutrients in food without changing the existing dietary pattern. So far, the research on oral iron supplements has gone through four stages: inorganic iron salts, organic iron salts, amino acid chelated iron, and antioxidant peptide chelated iron. Currently, the iron supplements widely used are mostly first- and second-generation iron supplements such as inorganic iron salts and organic iron salts. These traditional iron supplements mostly use inorganic iron such as ferrous sulfate or organic acid iron salts such as ferrous citrate and ferrous gluconate. Free iron ions have many disadvantages, such as poor bioavailability, poor stability, obvious gastrointestinal irritation and easy to cause adverse reactions, and the generation of endogenous free radicals. In order to overcome the many shortcomings of the first and second generation oral iron supplements such as low bioavailability, and to avoid the high cost of the third generation of amino acid chelated iron, many researchers have turned their attention to the research of peptide chelated iron.

[0004] The preparation of peptide iron chelates usually refers to the process of chelation reaction between inorganic iron ions and polypeptides with iron chelating activity produced by protein hydrolysis. For hemoglobin peptide iron chelates, the most commonly used sources of inorganic iron ions are FeCl2, FeSO4 or (NH4)2Fe(SO4)2, etc. The acquisition of blood protein peptides mainly depends on enzymatic hydrolysis. This process uses a broad spectrum of endogenous proteases to hydrolyze hemoglobin, and then acid precipitation is performed to separate the heme-containing part. Enzymatic hydrolysis has the advantages of mild reaction conditions, strong enzyme specificity, and controllable enzymatic hydrolysis process. However, in the current preparation process of blood peptide iron chelates, iron ions are added to the reaction system additionally, and the iron ions contained in heme are removed with acid treatment, and the endogenous iron is not utilized. Summary of the invention

[0005] Based on this, the present invention provides a one-pot prepared sheep blood phosphopeptide chelated iron, a preparation method and an application thereof, so as to solve the problem that endogenous iron is not utilized in the preparation process of peptide chelated iron in the prior art.

[0006] The present invention is achieved through the following technical solutions:

[0007] A one-pot method for preparing sheep blood phosphopeptide chelated iron comprises:

[0008] Dissolve sheep blood powder in water, wherein the mass volume ratio of the sheep blood powder to water is 5g-15g:100mL, and preheat to 50°C-55°C to obtain a sheep blood powder solution.

[0009] Papain is added to a sheep blood powder solution for enzymolysis to obtain a sheep blood peptide solution, and sodium pyrophosphate is added to the sheep blood peptide solution for phosphorylation modification to obtain a sheep blood phosphopeptide reaction solution; the mass volume ratio of the papain to the sheep blood powder solution is 1g-2g:100mL; the volume mass ratio of the sheep blood peptide solution to sodium pyrophosphate is 5mL-20mL:1g.

[0010] The pH value of the sheep blood phosphopeptide reaction solution was adjusted to 3.0-4.0 to dissociate the heme group to obtain a dissociation solution.

[0011] The dissociation solution was centrifuged, and the supernatant was recovered to obtain sheep blood phosphopeptide chelated iron.

[0012] Preferably, the volume mass ratio of the sheep blood peptide solution to sodium pyrophosphate is 5 mL:1 g.

[0013] Preferably, the enzymatic activity of the papain is 700,000 U / g to 800,000 U / g.

[0014] Preferably, the enzymolysis temperature is 50°C to 55°C, and the enzymolysis time is 5h to 6h.

[0015] Preferably, the stirring speed during the enzymatic hydrolysis is 200 rpm to 400 rpm.

[0016] Preferably, the specific steps of the phosphorylation modification are: adding sodium pyrophosphate to the sheep blood peptide solution, stirring at 50°C to 55°C for 1h to 2h, and then heat treating in 100°C boiling water for 10min to 15min to terminate the reaction.

[0017] Preferably, the stirring speed is 200 rpm to 400 rpm.

[0018] Preferably, hydrochloric acid is used to adjust the pH value of the sheep blood phosphopeptide reaction solution, and the concentration of the hydrochloric acid is 2 mol / L to 3 mol / L.

[0019] Preferably, the centrifugation temperature is 4°C to 5°C, and the centrifugation time is 10 min to 15 min.

[0020] The sheep blood phosphopeptide chelated iron is used in a biological peptide type iron supplement.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The present invention focuses on the reaction of enzymatic hydrolysis coordinated with phosphorylation modification and iron chelation, and simplifies the current complex preparation process of blood peptide iron chelate by using the iron ions (endogenous iron) contained in heme. It also provides a theoretical basis for the preparation of a new type of biological peptide iron supplement using sheep blood, and also provides new ideas for the high value of mutton industry by-products in the future.

[0023] (2) The present invention uses sheep blood as a raw material, hydrolyzes it with papain to prepare a sheep blood peptide solution, and improves its iron chelating properties through phosphorylation modification, so that it chelates with heme iron to prepare sheep blood phosphopeptide chelated iron. The prepared sheep blood phosphopeptide chelated iron has a phosphorylation degree of 8.44 g / L and has the highest iron chelation rate of 64.67%.

[0024] (3) The present invention characterizes the structure of the obtained sheep blood phosphopeptide chelated iron. The present invention successfully characterizes the structure of the sheep blood phosphopeptide chelated iron through particle size analysis, scanning electron microscopy and X-ray photoelectron spectroscopy results, indicating that iron ions are embedded in the surface of the sheep blood peptide solution after chelation to form a large number of chelates; X-ray photoelectron spectroscopy results show that the content of Fe and P elements in the sheep blood phosphopeptide chelated iron are increased, further proving the formation of sheep blood phosphopeptide chelated iron and the improvement of the iron chelating ability of sheep blood peptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 A technical roadmap in an embodiment of the present invention;

[0027] Figure 2 This is a graph showing the change in hydrolysis degree during the enzymatic hydrolysis of sheep blood in an embodiment of the present invention;

[0028] Figure 3 This is a graph showing the phosphorus content of sheep blood peptide solution and sheep blood phosphopeptide chelated iron in an embodiment of the present invention;

[0029] Figure 4This is a graph showing the change in iron content and iron chelation rate of sheep blood peptide solution and sheep blood phosphopeptide chelated iron in an embodiment of the present invention;

[0030] Figure 5 The ultraviolet absorption spectra of the sheep blood peptide solution and the sheep blood phosphopeptide chelated iron in the embodiments of the present invention are shown;

[0031] Figure 6 It is a fluorescence spectrum diagram of the sheep blood peptide solution and the sheep blood phosphopeptide chelated iron in the embodiment of the present invention;

[0032] Figure 7 This is a particle size diagram of the sheep blood peptide solution and the sheep blood phosphopeptide chelated iron in the embodiment of the present invention;

[0033] Figure 8 It is an infrared spectrum of the sheep blood peptide solution and the sheep blood phosphopeptide chelated iron in the embodiment of the present invention;

[0034] Fig. 9 The scanning electron microscope images of the sheep blood peptide solution and the sheep blood phosphopeptide chelated iron in the embodiment of the present invention, the left side is the scanning electron microscope image of the sheep blood peptide solution, and the right side is the scanning electron microscope image of the sheep blood phosphopeptide chelated iron, and the magnifications are 100×, 500× and 30000× from top to bottom respectively;

[0035] Fig.10 It is an X-ray photoelectron spectrum of the sheep blood peptide solution and the sheep blood phosphopeptide chelated iron in the embodiment of the present invention;

[0036] Fig.10 In the figure, a is the X-ray photoelectron spectrum of sheep blood peptide solution, and b is the X-ray photoelectron spectrum of sheep blood phosphopeptide chelated with iron. DETAILED DESCRIPTION

[0037] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are provided. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0039] (1) Materials and reagents involved in the embodiments of the present invention:

[0040] Table 1 Materials and reagents

[0041] Reagent name Specification Manufacturer Sheep Blood Meal Food Grade Shandong Huachen Biotechnology Co., Ltd. BP (Sheep Blood Peptide Solution) self made Laboratory-made Fe-P-BP (Sheep Blood Phospho-Peptide Chelated Iron) self made Laboratory-made Papain AR Beijing Solable Technology Co., Ltd. Sodium pyrophosphate AR Chongqing Chuandong Chemical Co., Ltd. Sodium hydroxide AR Chengdu Kelong Chemicals Co., Ltd. hydrochloric acid AR Chengdu Kelong Chemicals Co., Ltd. OPA (O-phthalaldehyde) AR Shanghai Tengzhun Biotechnology Co., Ltd. Methanol AR Chengdu Kelong Chemicals Co., Ltd. SDS (Sodium dodecyl sulfate) AR Guangzhou Saiguo Biotechnology Co., Ltd. Boric acid AR Chengdu Kelong Chemicals Co., Ltd. N-Acetyl-L-Cysteine AR Chengdu Kelong Chemicals Co., Ltd. L-Leucine AR Merck Chemical Technology (Shanghai) Co., Ltd.

[0042] (2) Instruments and equipment involved in the embodiments of the present invention:

[0043] Table 2 Instruments and equipment

[0044]

[0045] (3) Statistical analysis of data involved in the embodiments of the present invention

[0046] Microsoft 2019 software was used for data analysis. The experimental results were expressed as "mean ± standard deviation". SPSS22.0 statistical software was used for one-way ANOVA analysis and post hoc multiple comparisons. Duncan analysis was used to determine whether there were significant differences between the groups. P < 0.05 was considered significant. SigmaPlot 14.0 software was used for plotting to visualize the analysis results.

[0047] The inventive concept of the present invention is as follows:

[0048] At present, the research and development of iron nutritional supplements focuses on the chelation pathway of iron, and peptide iron chelates are one of the potential candidates. However, the low chelation rate of peptide chelated iron limits its wide application in the food industry, and its exact chelate structure has not yet been fully elucidated. To overcome this obstacle, phosphorylation modification of peptides is considered to be a strategy to improve the chelation ability of peptide iron. On the other hand, sheep blood, as one of the by-products produced in large quantities during mutton processing, is rich in nutrients such as protein and heme iron. It can be used as a raw material for iron supplements to prepare sheep blood peptide chelated iron, that is, the product obtained by enzymatic hydrolysis of sheep blood protein and chelation of iron salts, which has the high absorption rate of animal-derived heme iron and the high stability of peptide iron chelates. However, the peptide iron chelates currently developed from animal blood all introduce additional iron ions, ignoring the fact that heme itself contains iron ions. Therefore, it is necessary to explore strategies to achieve chelation of animal blood peptides with endogenous heme iron. Another industry problem lies in the complexity of the process. The preparation of peptide-iron chelates requires steps such as enzymatic hydrolysis, addition of iron sources for chelation, and ultrafiltration purification. If endogenous iron can be directly utilized, the above process can be simplified or even completed in one step, but no relevant reports have been seen so far.

[0049] Based on this, the present invention provides a one-pot method for preparing sheep blood phosphopeptide chelated iron, wherein a sheep blood peptide solution is prepared by hydrolysis with papain, and its iron chelating properties are improved by phosphorylation modification, so that it is chelated with heme iron to prepare sheep blood phosphopeptide chelated iron. Subsequently, the obtained sheep blood phosphopeptide chelated iron is structurally characterized to prove that we have solved the problem of the complex preparation process of the current blood peptide iron chelate and realized the utilization of the iron ions (endogenous iron) contained in heme. The specific technical roadmap of the present invention is as follows: Figure 1As shown, it provides a theoretical basis for the preparation of a new type of bio-peptide iron supplement using sheep blood, and also provides new ideas for the high value of mutton industry by-products in the future.

[0050] In order to better illustrate the present invention, the present invention is further described below in conjunction with the embodiments. The following are specific embodiments.

[0051] Sheep blood peptide solution, referred to as BP; sheep blood phosphopeptide reaction solution, referred to as P-BP; sheep blood phosphopeptide chelated iron, referred to as Fe-P-BP.

[0052] Determination of protein content and amino acid composition of sheep blood meal:

[0053] Refer to the national standard GB5009.5-2016 to determine the protein content of sheep blood meal. Weigh 0.3g of sample, 0.4g of copper sulfate solid and 6.0g of potassium sulfate solid and 20.0mL of sulfuric acid solution into a digestion tube, and then put it into a digestion furnace for digestion; after digestion, take it out and cool it, add 50.0mL of water, and perform liquid addition, distillation and titration on an automatic Kjeldahl nitrogen analyzer, and record the data. The protein content in the sample is calculated according to the following formula:

[0054]

[0055] Where: V1 is the volume of sulfuric acid standard solution consumed by the sample during titration, unit: mL; V2 is the volume of sulfuric acid standard solution consumed by the blank reagent during titration, unit: mL; c is 0.1 mol / L; m is the mass of the sample, unit: g; V3 is the volume of the absorbed digestion solution, unit: mL; F is taken as 5.7.

[0056] According to the national standard GB5009.124-2016, the amino acid composition of sheep blood meal was analyzed using an amino acid automatic analyzer. 0.2 g of sample was accurately weighed, and 6 mol / L HCl was added for hydrolysis for 24 h. After treatment, sodium citrate buffer solution was added to dissolve, and the sample was transferred to an injection bottle and entered into the amino acid automatic analyzer for determination.

[0057] The results showed that protein content and amino acid composition affect the metal binding ability of polypeptides. Analysis of the protein content and amino acid composition of sheep blood meal will reflect its possibility as a good ligand for iron ions. The protein content of sheep blood meal was determined to be 88.2g / 100g, and a total of 17 amino acids were detected. The specific amino acid composition percentages are shown in Table 3. Among them, the content of amino acids such as leucine, alanine, aspartic acid, and valine is relatively high, while relatively few amino acids include methionine, isoleucine, cysteine, etc., which are in line with the amino acid composition characteristics of typical livestock blood proteins.

[0058] In addition, glutamic acid, lysine and phenylalanine are also commonly reported as amino acids that are more abundant in blood proteins. Studies have shown that the ability of protein hydrolysates to chelate metals depends on acidic amino acids, such as glutamic acid, aspartic acid, and basic amino acid residues, such as lysine residues and arginine residues. At physiological pH, proteins or polypeptides containing a large amount of acidic amino acids such as glutamic acid and aspartic acid can increase the solubility of iron ions due to their negative charge, and these amino acids have a high affinity for forming coordination bonds with metal cations, which can give polypeptides good iron binding ability. Basic amino acids such as lysine can act as a covalent intermediate to bind metal cofactors to proteins or polypeptides. The content of these amino acids in sheep blood proteins is relatively high, so it can be inferred that sheep blood protein peptides obtained by hydrolysis of sheep blood powder may have a strong iron binding ability.

[0059] Table 3 Amino acid composition of sheep blood protein

[0060] Amino Acids content(%) Amino Acids content(%) Aspartic acid 10.25 Isoleucine 0.66 Threonine 4.69 Leucine 12.51 Serine 5.75 Leucine 1.78 Glutamate 7.34 Phenylalanine 5.52 Glycine 7.42 Lysine 7.45 Alanine 11.34 Histidine 5.23 Cysteine 0.36 Arginine 2.83 Valine 9.26 Proline 6.59 Methionine 1.04 / /

[0061] Note: In Table 3, / indicates that the cell has no corresponding data.

[0062] Determination of enzymatic hydrolysis capacity of sheep blood meal:

[0063] (1) Weigh 15 g of sheep blood powder and dissolve it in 100 mL of distilled water at a mass volume ratio of 10%. Place it in a constant temperature water bath and preheat it to 50°C. Add 1.65 g of papain at a mass ratio of 1%, and perform enzymolysis for 6 hours in a constant temperature reactor at 50°C and under continuous stirring. The stirring speed is 400 rpm. After the reaction is completed, heat treat it in a boiling water bath at 100°C for 15 minutes to terminate the reaction and cool it to room temperature. Use hydrochloric acid with a concentration of 2 mol / L to reduce the pH to 4.0 to dissociate the heme group. Centrifuge the obtained 10,000 g material at 4°C for 10 minutes, recover the supernatant containing the sheep blood peptide solution, and discard the precipitate.

[0064] (2) Determination of hydrolysis degree

[0065] The free amino groups of the sheep blood peptide solution were determined by the o-phthalaldehyde method, abbreviated as OPA, and the hydrolysis degree of the sheep blood powder was calculated. The o-phthalaldehyde reagent consists of 10mL, 50mM o-phthalaldehyde, 10mL, 50mM N-acetyl-L-cysteine, 5mL, 20% sodium dodecyl sulfate by mass volume ratio and 75mM, 100mM, pH = 9.5 borate buffer. The prepared o-phthalaldehyde reagent was stored in the dark for 1h before use. Accurately mix 10μL of sample or leucine standard with 1.2mL o-phthalaldehyde reagent and react at room temperature for 10min. The absorbance value was measured at 340nm by a UV-8000A model ultraviolet-visible spectrophotometer. The absorbance values ​​of a series of leucine solutions with known concentrations were measured by the above method, and a standard curve of concentration x and absorbance y was drawn y = 0.6019x + 0.0735, R 2 =0.9999. Substitute the absorbance value of the sample into the standard curve to obtain the concentration of free amino groups. The degree of hydrolysis of sheep blood powder, i.e. DH, is calculated as follows:

[0066]

[0067] In the formula: [NH2]1 is the free amino acid concentration in the sheep blood meal hydrolyzate, unit is mmol / mL; V4 is the volume of the sheep blood meal hydrolyzate, unit is mL; [NH2]2 is the free amino acid concentration in the completely hydrolyzed sheep blood meal solution, unit is mmol / mL; V5 is the volume of the completely hydrolyzed sheep blood meal solution, unit is mL.

[0068] The determination method of [NH2]2 is as follows: accurately weigh 0.2g of sheep blood powder sample into a hydrolysis tube, add 15mL of 6mol / L hydrochloric acid solution, repeat vacuuming 3 times, seal the tube and place it in an electric heating blast thermostat at 110℃ for 24h. Filter and wash, then transfer the washing liquid into a 50mL volumetric flask to make up the volume and determine its absorbance at 340nm using the o-phthalaldehyde method. Calculate the total free amino concentration in the sample based on the standard curve.

[0069] The results showed that the degree of protein hydrolysis was related to the free amino acid content. As the degree of hydrolysis increased, the peptide bonds in the macromolecular protein continued to break, releasing small molecular free amino acids, which increased the free amino acid content. Therefore, the free amino acid content at different enzymatic hydrolysis times determined by the o-phthalaldehyde method can be used to reflect the degree of hydrolysis of sheep blood protein and the changing trend of the enzymatic hydrolysis rate with the action time of papain. Leucine was used as the standard curve, and the free amino acid content was calculated according to the standard curve, and the regression equation was obtained: y = 0.6019x + 0.0735, R 2 =0.9999, and the hydrolysis degree of the sheep blood peptide solution during the enzymatic hydrolysis of sheep blood was calculated. The results are as follows Figure 2 shown.

[0070] As the enzymatic hydrolysis time increases, the free amino acid content continues to increase, that is, the hydrolysis degree of sheep blood protein continues to increase. When the enzymatic hydrolysis time is 6h, the free amino acid content reaches the highest, 0.6449μg / mL, P<0.05, and the hydrolysis degree of sheep blood protein is 14.40%. At the same time, the hydrolysis rate decreases slightly with the increase of hydrolysis time. The hydrolysis degree increases rapidly within the first 4h of hydrolysis, and the rising rate gradually slows down between 4h and 6h. This gradually slowing trend may be caused by reduced enzyme activity, reduced enzyme substrates, or product inhibition. During the hydrolysis process, as the enzyme action time increases, the substrate is continuously cut and decomposed, and the number of peptide bonds in the substrate that are sensitive to enzyme hydrolysis gradually decreases, resulting in a decrease in the hydrolysis rate. The above results show that papain can be used to compare the degree of improvement in the recovery rate of heme iron before and after phosphorylation modification of sheep blood powder.

[0071] Example 1: One-pot method for preparing sheep blood phosphopeptide chelated iron

[0072] Dissolve the sheep blood powder in distilled water, place it in a constant temperature water bath and preheat it to 50°C to obtain a sheep blood powder solution, and the mass volume ratio of the sheep blood powder to distilled water is 5g:100mL. Add papain with an enzyme activity of 700,000 U / g to the sheep blood powder solution, and perform enzymolysis for 5h in a constant temperature reactor at 50°C and under continuous stirring, and the stirring speed is 200rpm. Without centrifugation, a sheep blood peptide solution is obtained, and the mass volume ratio of the papain to the sheep blood powder solution is 1g:100mL. Sodium pyrophosphate is added to the sheep blood peptide solution and placed in a constant temperature reactor, and continued to stir for 1h at 50°C for phosphorylation modification, and the stirring speed is 200rpm. Heat treatment in a 100°C boiling water bath for 10min terminates the reaction, and cools to room temperature to obtain a sheep blood phosphopeptide reaction solution, and the volume mass ratio of the sheep blood peptide solution to sodium pyrophosphate is 5mL:1g. The pH of the sheep blood phosphopeptide reaction solution was lowered to 3.0 using hydrochloric acid at a concentration of 2 mol / L to dissociate the heme group to obtain a dissociation solution. The dissociation solution was centrifuged at 4°C for 10 minutes, the supernatant was recovered, and the precipitate was discarded to obtain sheep blood phosphopeptide chelated iron.

[0073] Example 2: One-pot method for preparing sheep blood phosphopeptide chelated iron

[0074] Dissolve the sheep blood powder in distilled water, place it in a constant temperature water bath and preheat it to 55°C to obtain a sheep blood powder solution, and the mass volume ratio of the sheep blood powder to distilled water is 5g:100mL. Add papain with an enzyme activity of 800,000 U / g to the sheep blood powder solution, and perform enzymolysis for 6 hours in a constant temperature reactor at 55°C and under continuous stirring, and the stirring speed is 400rpm. Without centrifugation, a sheep blood peptide solution is obtained, and the mass volume ratio of the papain to the sheep blood powder solution is 1g:100mL. Sodium pyrophosphate is added to the sheep blood peptide solution and placed in a constant temperature reactor, and continued to stir for 2 hours at 55°C for phosphorylation modification, and the stirring speed is 400rpm. Heat treatment in a 100°C boiling water bath for 15min to terminate the reaction, cool to room temperature, and obtain a sheep blood phosphopeptide reaction solution, and the volume mass ratio of the sheep blood peptide solution to sodium pyrophosphate is 10mL:1g. The pH of the sheep blood phosphopeptide reaction solution was lowered to 4.0 using hydrochloric acid at a concentration of 3 mol / L to dissociate the heme group to obtain a dissociation solution. The dissociation solution was centrifuged at 5°C for 15 minutes, the supernatant was recovered, and the precipitate was discarded to obtain sheep blood phosphopeptide chelated iron.

[0075] Example 3: One-pot method for preparing sheep blood phosphopeptide chelated iron

[0076] Dissolve the sheep blood powder in distilled water, place it in a constant temperature water bath and preheat it to 50°C to obtain a sheep blood powder solution, wherein the mass volume ratio of the sheep blood powder to distilled water is 15g:100mL. Add papain with an enzyme activity of 700,000 U / g to the sheep blood powder solution, and perform enzymolysis for 5h in a constant temperature reactor at 55°C and under continuous stirring, with a stirring speed of 200rpm. Without centrifugation, a sheep blood peptide solution is obtained, wherein the mass volume ratio of the papain to the sheep blood powder solution is 2g:100mL. Sodium pyrophosphate is added to the sheep blood peptide solution and placed in a constant temperature reactor, and continued to stir for 1h at 50°C for phosphorylation modification, with a stirring speed of 200rpm. Heat treatment in a 100°C boiling water bath for 10min terminates the reaction, and cools to room temperature to obtain a sheep blood phosphopeptide reaction solution, wherein the volume mass ratio of the sheep blood peptide solution to sodium pyrophosphate is 15mL:1g. The pH of the sheep blood phosphopeptide reaction solution was lowered to 3.0 using hydrochloric acid at a concentration of 2 mol / L to dissociate the heme group to obtain a dissociation solution. The dissociation solution was centrifuged at 4°C for 10 minutes, the supernatant was recovered, and the precipitate was discarded to obtain sheep blood phosphopeptide chelated iron.

[0077] Example 4: One-pot method for preparing sheep blood phosphopeptide chelated iron

[0078] Dissolve the sheep blood powder in distilled water, place it in a constant temperature water bath and preheat it to 55°C to obtain a sheep blood powder solution, and the mass volume ratio of the sheep blood powder to distilled water is 15g:100mL. Add papain with an enzyme activity of 800,000 U / g to the sheep blood powder solution, and perform enzymolysis for 6h in a constant temperature reactor at 50°C and under continuous stirring, and the stirring speed is 400rpm. Without centrifugation, a sheep blood peptide solution is obtained, and the mass volume ratio of the papain to the sheep blood powder solution is 2g:100mL. Sodium pyrophosphate is added to the sheep blood peptide solution and placed in a constant temperature reactor, and continued to stir for 2h at 55°C for phosphorylation modification, and the stirring speed is 400rpm. Heat treatment in a 100°C boiling water bath for 15min to terminate the reaction, cool to room temperature, and obtain a sheep blood phosphopeptide reaction solution, and the volume mass ratio of the sheep blood peptide solution to sodium pyrophosphate is 20mL:1g. The pH of the sheep blood phosphopeptide reaction solution was lowered to 4.0 using hydrochloric acid at a concentration of 3 mol / L to dissociate the heme group to obtain a dissociation solution. The dissociation solution was centrifuged at 5°C for 15 minutes, the supernatant was recovered, and the precipitate was discarded to obtain sheep blood phosphopeptide chelated iron.

[0079] Comparative Example 1

[0080] In this comparative example, sodium pyrophosphate was not used to phosphorylate the sheep blood peptide solution, and the remaining steps were the same as the preparation method in the example.

[0081] Example 5: Evaluation of phosphorylation degree

[0082] The phosphorylation degree of the sheep blood phosphopeptide chelated iron prepared in Examples 1 to 4 and Comparative Example 1 was evaluated.

[0083] The evaluation index of phosphorylation degree is the phosphorus content in sheep blood phosphopeptide chelated iron. The determination method refers to the national standard GB5009.268-2016 and is carried out using an inductively coupled plasma emission spectrometer. Weigh 3.00 mL of the sheep blood phosphopeptide chelated iron liquid obtained in Example 1 to Example 4 and Comparative Example 1 respectively in a microwave digestion inner tank, add 5 mL of nitric acid, cover and place for 1 hour or overnight, tighten the tank cover and place it in a microwave digestion instrument for digestion for 15 minutes, and set the temperature to 150 ° C. After the digestion is completed, it is cooled, exhausted, and constant volume, and then injected into an inductively coupled plasma mass spectrometer, the plasma gas flow rate is 12.0 L / min, the auxiliary gas flow rate is 1.0 L / min, and the nebulizer carrier gas flow rate is 0.70 L / min. The signal response values ​​of the P element and the internal standard element are determined, and the concentration of the phosphorus element in the digestion solution is obtained according to the standard curve.

[0084] The phosphorus content of the sheep blood phosphopeptide chelated iron obtained in Examples 1 to 4 and Comparative Example 1 is as follows: Figure 3As shown. It can be seen that compared with Comparative Example 1, the phosphorus content of the sheep blood phosphopeptide chelated iron obtained in Examples 1 to 4 is increased. Among them, the phosphorus content of the sheep blood phosphopeptide chelated iron obtained in Example 1 increases most significantly, reaching 8.44 g / L, indicating that when the volume mass ratio of the sheep blood peptide solution to sodium pyrophosphate is 5:1, the phosphorylation degree of the sheep blood phosphopeptide chelated iron is optimal.

[0085] Example 6, determination of heme iron chelation rate

[0086] The heme iron chelation rate of the sheep blood phosphopeptide chelated iron prepared in Examples 1 to 4 and Comparative Example 1 was measured.

[0087] The heme iron chelation rate is expressed as the amount of heme iron released by hydrolysis, that is, the ratio between the amount chelated in the supernatant of the soluble peptide and the iron content in the blood meal sample, and is calculated as follows:

[0088]

[0089] Wherein: [Fe]1 is the iron content chelated in the sheep blood peptide solution, in mg / L; [Fe]2 is the iron content of the sheep blood powder, in mg / L.

[0090] The determination was performed using an inductively coupled plasma emission spectrometer. 3.00 mL of the sheep blood peptide solution and 0.5 g of the sheep blood powder sample in Examples 1 to 4 and Comparative Example 1 were weighed separately in a microwave digestion inner tank, 5 mL of nitric acid was added, the cover was placed for 1 hour, the tank cover was tightened and placed in a microwave digestion instrument for digestion for 15 minutes, and the temperature was set to 150 ° C. After the digestion was completed, it was cooled, exhausted, and constant in volume, and then injected into an inductively coupled plasma mass spectrometer, the plasma gas flow rate was 12.0 L / min, the auxiliary gas flow rate was 1.0 L / min, and the nebulizer carrier gas flow rate was 0.70 L / min. The signal response values ​​of the Fe element and the internal standard element were determined, and the concentration of the Fe element in the digestion solution was obtained according to the standard curve. The heme iron chelation rates of the sheep blood peptide solution and the sheep blood phosphopeptide chelated iron in Examples 1 to 4 and Comparative Example 1 were calculated respectively, and compared and analyzed.

[0091] The iron content in the sheep blood peptide solution and the sheep blood phosphopeptide chelated iron was measured respectively, and the iron chelation rate was calculated according to the iron content of the raw material, which can reflect the degree of improvement of the iron chelation ability of the sheep blood peptide by phosphorylation modification. Figure 4As shown, the iron content and iron chelation rate in the sheep blood peptide solution and the sheep blood phosphopeptide chelated iron are changed. It was observed that in Comparative Example 1, the iron content and iron chelation rate of the unmodified sheep blood peptide solution were the lowest, only 2.95 mg / L and 10.27%, P<0.05. This may be due to the limited hydrolysis to form a macromolecular insoluble polymer and hindered. After phosphorylation modification in Examples 1 to 4, it was observed that the iron content in the sheep blood phosphopeptide chelated iron was greatly increased. Among them, the iron content and iron chelation rate in Example 1 reached a maximum of 18.58 mg / L and 64.67%, P<0.05. And with the increase in the amount of sodium pyrophosphate, the iron content and iron chelation rate in the sheep blood phosphopeptide chelated iron maintained an increasing trend, which means that its iron chelation properties continue to improve. At present, similar studies have measured the chelation rate after adding metal ions, and no reports have been reported on the comparison of endogenous metal ion chelation rates before and after modification. After comparison, the iron chelating ability of phosphorylated sheep blood phosphopeptide chelated iron modified by the volume mass ratio of sheep blood peptide solution to sodium pyrophosphate of 5:1 was the best, and the iron chelating rate reached 64.67%, which was not lower than the iron chelating active peptides purified and separated from other protein hydrolysates.

[0092] Example 7: Structural characterization of sheep blood peptide solution and sheep blood phosphopeptide chelated iron

[0093] The sheep blood peptide solutions and sheep blood phosphopeptide chelated iron in Examples 1 to 4 and Comparative Example 1 were subjected to ultraviolet-visible absorption spectroscopy analysis, abbreviated as UV-Vis.

[0094] The sheep blood peptide solution and sheep blood phosphopeptide chelated iron were diluted 20 times with distilled water, and the dry matter content was 5 mg / mL. The UV-visible spectrophotometer was used to scan in the wavelength range of 190nm to 450nm to analyze and compare the absorbance of each sample. Before measuring the sample, the UV-visible spectrophotometer was blank calibrated with distilled water.

[0095] UV-visible spectroscopy was used to determine the chelation of sheep blood peptide solution with iron ions after phosphorylation modification. The formation of complexes between organic ligands and metal ions will cause the movement of the original absorption peak or the appearance or disappearance of new absorption peaks, which is widely used to prove the formation of peptide iron chelates. Figure 5 This is the ultraviolet absorption spectrum of sheep blood peptide solution and sheep blood phosphopeptide chelated iron. It can be seen that both the sheep blood peptide solution and the sheep blood phosphopeptide chelated iron have many absorption peaks in the range of 190nm to 230nm, which is usually related to the carbonyl group and amide bond in the polypeptide.

[0096] Most studies on peptide iron chelates have observed a greater absorbance after chelation. However, in this study, it was observed that the absorbance of sheep blood phosphopeptide chelated iron was slightly lower than that of sheep blood peptide solution, which is most likely due to the effect of phosphorylation modification on the structure of the peptide. It can be clearly seen in the figure that as the degree of phosphorylation increases, the trend of decreasing absorbance becomes more and more obvious. This is because the esterification reaction between phosphate ions and amino acid residues forms a network structure, which reduces the exposure of amino acid residues and thus reduces their absorbance. In addition to the sheep blood phosphopeptide chelated iron modified with sodium pyrophosphate at a volume mass ratio of 20:1 to the sheep blood peptide solution, the absorbance has not yet dropped significantly due to the low degree of phosphorylation. The overall absorbance shows a trend of increasing relative to the sheep blood peptide solution, which proves the formation of sheep blood phosphopeptide chelated iron, that is, the chelation between the peptide and the iron ion.

[0097] Example 8: Structural characterization of sheep blood peptide solution and sheep blood phosphopeptide chelated iron

[0098] Fluorescence spectroscopy analysis of the sheep blood peptide solution and the sheep blood phosphopeptide chelated iron in Examples 1 to 4 and Comparative Example 1

[0099] The sheep blood peptide solution and sheep blood phosphopeptide chelated iron were diluted 20 times with distilled water, and the dry matter content was 5 mg / mL. The fluorescence spectrum of each sample was measured by a fluorescence spectrophotometer. The excitation wavelength was fixed at 275 nm, and the emission wavelength range was selected to be 300 nm.

[0100] Fluorescence spectroscopy was used to study the chelation of sheep blood peptide solution with iron ions after phosphorylation modification. In the fluorescence spectrum of protein hydrolysates or peptides, phenylalanine, with a maximum absorption wavelength of 280nm, tyrosine and tryptophan residues as natural chromophores can emit fluorescence. When metals chelate with peptides containing these amino acids, it will lead to a change in the maximum emission wavelength or a decrease in the peak intensity, which can be used to characterize metal-peptide chelates. Figure 6 Shown are the fluorescence spectra of sheep blood peptide solution and sheep blood phosphopeptide chelated iron. The results show that when the sheep blood peptide solution is excited at 275nm, phenylalanine produces the maximum endogenous fluorescence emission at 280nm. The fluorescence intensity of the phosphorylated sheep blood phosphopeptide chelated iron is lower than that of the sheep blood peptide solution, and the fluorescence intensity decreases significantly with the increase of the phosphorylation degree.

[0101] It is reported that the decrease in fluorescence intensity is a typical phenomenon after peptide folding, which indicates that iron ions may bind to the chelation site through coordination bonds to produce a folded structure, that is, sheep blood phosphopeptide chelated iron is generated. During the phosphorylation process, the strong steric hindrance effect generated by the phosphorus group will also shield the fluorescent residues, resulting in a decrease in fluorescence intensity. High concentrations of iron may also cause fluorescence quenching, further leading to a decrease in fluorescence intensity. In addition, it was observed that the maximum emission wavelength of sheep blood phosphopeptide chelated iron after phosphorylation modification was slightly blue-shifted from 280nm to 278nm, which may be due to the increase in the hydrophobicity of the protein molecule surface caused by phosphorylation. The addition of metal ions may also transfer amino acids to the molecular surface, resulting in the movement of the maximum absorption peak, further illustrating the formation of sheep blood phosphopeptide chelated iron.

[0102] Combined with the results of phosphorylation degree, iron chelation rate, UV-visible spectrum and fluorescence spectrum, it was found that the sheep blood phosphopeptide chelated iron obtained by phosphorylation modification with a volume mass ratio of 5:1 of sheep blood peptide solution and sodium pyrophosphate in Example 1 had the best chelation effect. The changes in various indicators were obvious and typical, so the sample was selected for the following structural characterization analysis.

[0103] Example 9: Structural characterization of sheep blood peptide solution and sheep blood phosphopeptide chelated iron

[0104] In this example, the particle size analysis of sheep blood peptide solution and sheep blood phosphopeptide chelated iron was performed.

[0105] The sheep blood peptide solution and sheep blood phosphopeptide chelated iron were diluted 20 times with distilled water, the dry matter was 5 mg / mL, and filtered through a 0.22 μm filter membrane. The particle size distribution of the samples was determined by a nanoparticle size analyzer at 25°C. Each sample was tested in triplicate, and the experimental results were expressed as the average particle size.

[0106] The particle size of sheep blood protein peptide before and after the chelation reaction with iron ions can be reflected by the particle size. The particle size analysis results of sheep blood phosphopeptide chelated iron prepared by 3.0g sodium pyrophosphate modification and unmodified sheep blood peptide solution are shown in Figure 7 The chelation of minerals and polypeptides includes both intramolecular and intermolecular interactions. The main reason may be that the chelate bonds formed between metal ions and polypeptides cross-link the polypeptides to form larger molecules. At the same time, the introduction of phosphate groups also increases the size of polypeptide particles. Figure 7 The average particle size of sheep blood phosphopeptide chelated iron was 712.7 nm, which was significantly larger than the average particle size of sheep blood peptide solution of 367.9 nm. This indicated that phosphorylation modification and iron chelation reaction led to an increase in the average particle size of sheep blood peptide solution particles, proving the formation of peptide-iron chelate.

[0107] Example 10: Structural characterization of sheep blood peptide solution and sheep blood phosphopeptide chelated iron

[0108] In this example, the sheep blood peptide solution and the sheep blood phosphopeptide chelated iron were subjected to Fourier transform infrared spectroscopy analysis, abbreviated as FTIR.

[0109] After the sheep blood peptide solution and sheep blood phosphopeptide chelated iron were freeze-dried, 2 mg of each sample was placed in a mortar, and an appropriate amount of potassium bromide powder was added. The mixture was mixed and ground under infrared light to make it uniform, and then placed in a tableting mold to obtain a sample slice. The transmittance was measured by scanning with a Fourier transform infrared spectrometer, and the wave number range was 400 cm -1 ~4000cm -1 .

[0110] Infrared spectroscopy was used to analyze the changes in the spatial structure of the sheep blood peptide solution after phosphorylation and the coordination reaction between metal ions and organic groups during the formation of sheep blood phosphopeptide chelated iron. Figure 8 The infrared spectra of sheep blood peptide solution and sheep blood phosphopeptide chelated iron are shown. It can be observed that the infrared spectra of sheep blood peptide solution and sheep blood phosphopeptide chelated iron before and after phosphorylation are 3406.38cm -1 The absorption peak at 3414.38 cm-1 was blue-shifted to 3414.38 cm-1 after phosphorylation. -1 This is due to the stretching vibration of OH and NH, indicating that the introduced phosphate group is connected with the hydroxyl oxygen and amino nitrogen. After phosphorylation, the fishbone collagen peptide is at 3369.08 cm -1 The absorption peak at blue shifted to 3403.80 cm -1 In addition, at 1078.80cm -1 A new absorption peak was observed at 1100 cm-1 for the phosphopeptide chelated iron in sheep blood. -1 ~920cm -1 This indicates that the phosphate group successfully binds to the sheep blood peptide.

[0111] Analysis of the formation mechanism of peptide iron chelate, 3406.38cm -1 To 3414.38cm -1 The blue shift of the absorption peak may also represent that NH is involved in the formation of peptide-iron chelate. According to the study of Fan et al., the infrared spectra of sea cucumber peptide and sea cucumber peptide chelated iron were compared and it was found that after the peptide chelated with iron ions, the NH stretching vibration band changed from 3401cm to 4401cm. -1 Blue shift to 3405cm -1 In addition, the spectrum of sheep blood phosphopeptide chelated iron is at 1651cm -1 The absorption peak of the amide I band is 1700 cm -1 ~1600cm -1, which belongs to the stretching vibration of C=O. This phenomenon shows that the vibration intensity of C=O in the sheep blood phosphopeptide chelated iron increases, the hydrogen bonding effect in the chelate molecule weakens, and more C=O provides coordination for iron ions.

[0112] In addition, it was noted that sheep blood phosphopeptide chelated iron at 900 cm -1 ~500cm -1 Several absorption peaks were generated in the range, which were not found in the spectrum of the sheep blood peptide solution. This is caused by the vibration of CH and NH in the iron ion chelating peptide, which is consistent with the peptide iron chelate data from other sources. The movement of the vibration bands in these characteristic regions can be inferred that the binding of the sheep blood peptide solution to Fe is carried out through -NH2 and COO- groups, indicating that the iron ions may chelate with these groups to form Fe-N or COO-Fe. This may be because the nitrogen atom can form a coordination bond with the iron ion by providing an electron pair, while the carbonyl oxygen has a non-bonded free electron pair to chelate the iron ion.

[0113] Example 11: Structural characterization of sheep blood peptide solution and sheep blood phosphopeptide chelated iron

[0114] In this example, the sheep blood peptide solution and the sheep blood phosphopeptide chelated iron were subjected to scanning electron microscopy, abbreviated as SEM.

[0115] The prepared sheep blood peptide solution and sheep blood phosphopeptide chelated iron powder were evenly coated on the sample plate with double-sided adhesive, and then gold-sprayed. Scanning electron microscope was used to obtain scanning images of the sample at 100×, 500× and 30000× magnifications at an accelerating voltage of 1 kV and a working distance of 3.8 mm.

[0116] Scanning electron microscopy was used to visually describe the microstructure of the chelate. Fig. 9 The figure shows the scanning electron microscope images of sheep blood peptide solution and sheep blood phosphopeptide chelated iron observed at 100 times, 500 times and 30,000 times respectively. Although there are slight pits on the surface of the sheep blood peptide solution particles, which may be caused by freeze drying or conductive glue, it is generally smooth and has a plate-like lamellar structure. At 30,000 times, its surface is flat, dense and free of particles. The sheep blood phosphopeptide chelated iron shows a loose flocculent structure, the plate-like structure is destroyed to produce irregular lobes, the surface becomes obviously rough, and many granular aggregates appear.

[0117] This phenomenon shows that sheep blood peptide solution and sheep blood phosphopeptide chelated iron are two different substances, and the same changes have been observed in many recent studies. Phosphorylation modification may cause changes in the surface properties of sheep blood peptide solution, such as exposing new binding sites or changes in surface charge, which explains why the smooth plate-like structure on the surface of the chelate is irregular. At the same time, the crystalline particles on the surface of the mineral chelate may be produced by the combination of peptides and mineral ions. The embedding of iron ions destroys the original dense structure of the sheep blood peptide solution, resulting in the generation of more holes, thereby changing the properties of the peptide. This further confirms the formation of sheep blood phosphopeptide chelated iron.

[0118] Example 12: Structural characterization of sheep blood peptide solution and sheep blood phosphopeptide chelated iron

[0119] In this example, X-ray photoelectron spectroscopy, abbreviated as XPS, was performed on the sheep blood peptide solution and the sheep blood phosphopeptide chelated iron.

[0120] Add gold palladium to the prepared sheep blood peptide solution and sheep blood phosphopeptide chelated iron powder, and place them in the X-ray spectrometer analysis room for analysis. Determine the relative concentrations of C, Fe, N, O and P elements in the samples, and use Avantage software for data analysis.

[0121] X-ray photoelectron spectroscopy is a method that uses an electron spectrometer to measure the electron binding energy of photoelectrons emitted from the surface of a sample when it is irradiated with X-ray photons, thereby reflecting the chemical composition. It can be used to determine the chemical element composition, chemical state, and molecular structure of the solid surface. The content or concentration of elements on the sample surface can be obtained based on the peak intensity of the spectrum. Fig.10 X-ray photoelectron spectra of sheep blood peptide solution and sheep blood phosphopeptide chelated iron. Fig.10 a in the equation is the energy spectrum of sheep blood peptide solution. Fig.10 b is the energy spectrum of sheep blood phosphopeptide chelated iron. Table 4 lists the percentage of C, N, O, Fe and P elements on the surface of sheep blood peptide solution and sheep blood phosphopeptide chelated iron. The main elements in the sheep blood peptide solution are C, O, and N, with atomic percentages of 62.72%, 22.89% and 13.68%, respectively. In addition, it also contains very small amounts of Fe and P. After phosphorylation modification, the atomic percentage of P in sheep blood phosphopeptide chelated iron increased from 0.35% to 2.74%, indicating that phosphate was introduced into the peptide.

[0122] In addition, the atomic percentage of Fe increased from 0.36% to 0.59%, an increase of more than 1.5 times, indicating that the sheep blood peptide solution was chelated with iron ions to form a peptide-iron chelate, and proved that phosphorylation modification can enhance the iron chelation ability of the sheep blood peptide solution to a certain extent.

[0123] Table 4 Atomic percentage of C, N, O, Fe and P in sheep blood peptide solution and sheep blood phosphopeptide chelated iron

[0124] element% C N O Fe P Sheep blood peptide solution 62.72 13.68 22.89 0.36 0.35 Sheep blood phosphopeptide chelation 55.02 13.00 28.65 0.59 2.74

[0125] In summary, the present invention selects sheep blood as raw material, hydrolyzes sheep blood peptide solution by papain, and improves its iron chelating properties by phosphorylation modification, so that it is chelated with heme iron to prepare sheep blood phosphopeptide chelated iron. The prepared sheep blood phosphopeptide chelated iron has a phosphorylation degree of 8.44 g / L and has the highest iron chelation rate of 64.67%.

[0126] Subsequently, the obtained sheep blood phosphopeptide chelated iron was structurally characterized. The present invention illustrates the effect of phosphorylation on the structure of the sheep blood peptide solution and the main groups that may be involved in the chelation reaction through ultraviolet spectral analysis, fluorescence spectral analysis, and infrared spectral analysis results. The decrease in absorbance in the ultraviolet-visible spectrum indicates that the amino acid residues in the sheep blood peptide solution reacted with the phosphate groups during the phosphorylation process to produce a network structure; the decrease in fluorescence intensity indicates that the peptide may have folded during the peptide iron chelation process; the change in the infrared absorption peak can be inferred that the binding of the sheep blood peptide solution to Fe during the chelation process is mainly carried out through -NH2 and COO- groups, and the iron ions also react with the phosphate groups in the sheep blood peptide solution to form sheep blood phosphopeptide chelated iron.

[0127] In addition, the present invention successfully characterized the structure of sheep blood phosphopeptide chelated iron through particle size analysis, scanning electron microscopy and X-ray photoelectron spectroscopy results. Particle size analysis shows that the particle size of sheep blood phosphopeptide chelated iron is 712.7nm, which is larger than the particle size of sheep blood peptide solution 367.9nm, which reflects the introduction of phosphate groups and the formation of peptide iron chelates; scanning electron microscope images intuitively show the microscopic morphology of the surface of sheep blood peptide solution and sheep blood phosphopeptide chelated iron P, which changes from smooth to rough, and a large number of granular aggregates appear, indicating that the sheep blood peptide solution is embedded with iron ions on the surface after chelation, forming a large number of chelates; X-ray photoelectron spectroscopy results show that the content of Fe and P elements in sheep blood phosphopeptide chelated iron increases, further proving the formation of sheep blood phosphopeptide chelated iron and the improvement of sheep blood peptide iron chelating ability.

[0128] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0129] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A one-pot method for preparing sheep blood phosphopeptide chelated iron, characterized in that: include: Dissolve sheep blood powder in water, wherein the mass volume ratio of the sheep blood powder to water is 5 g to 15 g: 100 mL, and preheat to 50° C. to 55° C. to obtain a sheep blood powder solution; Adding papain to a sheep blood powder solution for enzymolysis to obtain a sheep blood peptide solution, and adding sodium pyrophosphate to the sheep blood peptide solution for phosphorylation modification to obtain a sheep blood phosphopeptide reaction solution; the mass volume ratio of the papain to the sheep blood powder solution is 1 g to 2 g: 100 mL; the volume mass ratio of the sheep blood peptide solution to sodium pyrophosphate is 5 mL to 20 mL: 1 g; The pH value of the sheep blood phosphopeptide reaction solution is adjusted to 3.0-4.0 to dissociate the heme group to obtain a dissociation solution; The dissociation solution was centrifuged, and the supernatant was recovered to obtain sheep blood phosphopeptide chelated iron.

2. The method for preparing sheep blood phosphopeptide chelated iron by one-pot method as claimed in claim 1, characterized in that: The volume mass ratio of the sheep blood peptide solution to sodium pyrophosphate is 5 mL:1 g.

3. The method for preparing sheep blood phosphopeptide chelated iron by one-pot method as claimed in claim 1, characterized in that: The enzymatic activity of the papain is 700,000 U / g to 800,000 U / g.

4. The method for preparing sheep blood phosphopeptide chelated iron by one-pot method as claimed in claim 1, characterized in that: The enzymolysis temperature is 50° C. to 55° C., and the enzymolysis time is 5 h to 6 h.

5. The method for preparing sheep blood phosphopeptide chelated iron by one-pot method as claimed in claim 1, characterized in that: The specific steps of the phosphorylation modification are: adding sodium pyrophosphate to the sheep blood peptide solution, stirring at 50°C to 55°C for 1h to 2h, and then heat-treating in 100°C boiling water for 10min to 15min to terminate the reaction.

6. The method for preparing sheep blood phosphopeptide chelated iron by one-pot method as claimed in claim 1, characterized in that: The pH value of the sheep blood phosphopeptide reaction solution was adjusted using hydrochloric acid, wherein the concentration of the hydrochloric acid was 2 mol / L to 3 mol / L.

7. The method for preparing sheep blood phosphopeptide chelated iron by one-pot method as claimed in claim 1, characterized in that: The centrifugation temperature is 4°C to 5°C, and the centrifugation time is 10min to 15min.

8. Sheep blood phosphopeptide chelated iron prepared by the method for preparing sheep blood phosphopeptide chelated iron by one-pot method as claimed in claim 1.

9. Use of the sheep blood phosphopeptide chelated iron as claimed in claim 8 in a biological peptide type iron supplement.

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