Food-borne supplement for preventing iron deficiency symptom

Abstract

The invention relates to a food-borne iron nutritional supplement, in particular to a quinoa protein peptide-iron chelate and a preparation method thereof. The chenopodium quinoa protein peptide-iron chelate is prepared by taking nutrient-rich chenopodium quinoa protein as a peptide source and inorganic iron salt as an iron source; the preparation method comprises the following steps: S1, extraction of quinoa protein; S2, inoculation and fermentation; S3, enzymolysis; S4, ultrafiltration; the preparation method is simple in technological process, high in production efficiency and suitable for industrial large-scale production; the content of small molecule peptide in the prepared peptide-iron chelate is up to 80% or above, the content of chelated iron ions is 10% or above, and the chelation rate reaches 98% or above; the prepared chenopodium quinoa protein peptide-iron chelate has relatively high stability in the gastric digestion process, and has smaller irritation to the stomach; the product is more easily digested and absorbed by a human body in intestinal tracts, and the iron supplementing effect is better; the traditional Chinese medicine composition is safe, free of toxic and side effects, free of allergy and wide in applicable crowd range.

Description

Technical Field

[0001] The invention relates to a food-derived iron nutritional supplement, and in particular to a quinoa protein peptide-iron chelate and a preparation method thereof. Background Art

[0002] Iron is one of the essential trace elements for the human body. It is an indispensable and important component of hemoglobin, myoglobin, cytochrome, enzymes, etc. It plays an important role in human metabolism. Iron deficiency is the most common nutritional disorder, common in the elderly and children. This is mainly due to insufficient dietary intake or insufficient absorption and utilization. In severe cases, it can lead to anemia, decreased immunity, mental retardation, neurodegeneration and other diseases.

[0003] Eating iron-fortified foods has now become an effective method for preventing and improving iron deficiency worldwide. The development of existing iron supplements has gone through four major stages: the first generation of iron supplements is inorganic iron salts, including ferrous sulfate, ferrous chloride, ferrous fumarate and other iron supplements. However, this type of iron supplement has poor stability, low absorption rate, and adverse reactions such as stomach irritation, long-term diarrhea, and nausea. The second generation of iron supplements is iron salt complexes represented by ferrous citrate and complexes such as ferrous lactate. These types of iron supplements are less irritating to the stomach and intestines, but their stability is also low. The third generation of iron supplements is amino acid chelated iron, including ferrous glycine and ferrous threonate. Amino acid chelated iron can be absorbed through the amino acid channel in the small intestine, and its utilization rate is significantly higher than that of the first and second generation iron supplements, and it avoids irritation to the gastrointestinal tract and has good stability, but due to its high cost, it cannot be promoted at present. The fourth generation of iron supplements includes heme iron, polysaccharide iron, and peptide iron. These iron supplements are non-irritating to the gastrointestinal tract and have good iron supplementation effects. Among them, iron chelate peptides, as a new type of iron supplement, have been reported to have better bioavailability and no side effects, lower cost and higher stability compared with third-generation iron supplements. Quinoa protein is rich in amino acids. In addition to the nine essential amino acids, it also contains a variety of non-essential amino acids, especially lysine and tryptophan, which are suitable for human absorption.

[0004] In the existing technology of peptide iron chelates, various forms of iron supplements have been developed, but there is still room for improvement. Compared with the research in the existing literature, we found that the peptides isolated from quinoa protein have a higher chelation rate with ferrous ions, stronger binding ability, and stronger resistance to digestion during gastrointestinal digestion. In addition, by optimizing the selection of peptides, we can obtain peptide iron chelates with better absorption in the small intestine, thereby overcoming the absorption problem of traditional iron supplements and improving bioavailability.

[0005] Quinoa protein peptide is a high-quality protein extracted from quinoa. It is rich in essential amino acids and its biological value is close to that of animal protein, which helps muscle recovery and growth. Compared with some plant proteins, quinoa protein peptide has a higher digestibility and is suitable for people who have difficulty digesting certain proteins. Quinoa protein peptide is a high-quality protein from plant sources, non-allergenic, and suitable for vegetarians and people who are allergic to dairy products or animal proteins. In addition, in preliminary experiments, we found that quinoa protein peptide has a good chelation rate with ferrous ions and has good potential for preparing peptide iron chelates.

[0006] Therefore, it is necessary to develop a quinoa protein peptide-iron chelate with good iron chelating effect and easy digestion and absorption. Summary of the invention

[0007] The purpose of the present invention is to propose a quinoa protein peptide-iron chelate and a preparation method thereof based on the above situation, which can be used as a food-derived food supplement for the elderly to prevent iron deficiency symptoms.

[0008] The first aspect of the present invention is to provide a method for preparing quinoa protein peptide-iron chelate, comprising the following steps:

[0009] S1. Extraction of quinoa protein: taking quinoa powder, adding solvent to extract and defatting, solid-liquid separation, drying, adding water, adjusting the pH value to 9-10, stirring evenly, filtering the precipitate, and obtaining a protein extract; adjusting the pH of the protein extract to the isoelectric point, centrifuging, washing, and drying to obtain quinoa protein;

[0010] S2. Inoculation and fermentation: dissolving the quinoa protein obtained in step S1 in water to prepare a quinoa protein solution, and inoculating lactic acid bacteria for fermentation;

[0011] S3. Enzymatic hydrolysis: Take the fermentation broth obtained in step S2, add protease, and perform enzymatic hydrolysis to obtain an enzymatic hydrolyzate.

[0012] S4. Ultrafiltration: Take the hydrolyzate obtained in step S3, and use a ceramic membrane of 2000Da and 5000Da to retain the hydrolyzate with a molecular weight cutoff of 0-2000Da, 2000Da-5000Da and>5000Da to obtain a quinoa protein peptide solution;

[0013] S5. Chelation reaction: Add a water-soluble iron salt to the quinoa protein peptide solution obtained in step S4, adjust the pH to 6.0-8.0, and stir the chelation reaction at 30°C-50°C to obtain a quinoa protein peptide-iron chelation reaction solution;

[0014] S6. Take the reaction solution obtained in step S5, collect the precipitate after alcohol precipitation to obtain quinoa protein peptide-iron chelate.

[0015] Further, in step S1, the extraction solvent is selected from n-hexane and deionized water; preferably n-hexane;

[0016] In some embodiments of the present invention, the ratio of quinoa flour to extraction solvent (w / v) is 1:4-1:12.

[0017] In some embodiments of the present invention, in step S1, the protein content of the quinoa protein powder is ≥ 85%.

[0018] In some embodiments of the present invention, in step S2, the lactic acid bacteria are selected from Lactobacillus plantarum, Lactobacillus bulgaricus and Streptococcus thermophilus;

[0019] Further, in step S2, the fermentation conditions are as follows: the ratio of water to quinoa protein is 1:20-1:40, and the inoculation concentration is 10 7 -10 8 CFU / mL, fermentation time is 24-48 hours.

[0020] In a preferred embodiment, the fermentation conditions are as follows: the ratio of water to quinoa protein is 1:40, and the fermentation time is 24 hours.

[0021] In some embodiments of the present invention, in step S3, the pH value of the quinoa protein solution is adjusted to 8.0-9.0, a protease is added, and after enzymolysis at 50°C-55°C for 3-4 hours, the temperature is raised to inactivate the enzyme to obtain an enzymolysis solution. In some embodiments, the enzymolysis solution is heated to 95°C to inactivate the enzyme for 10 minutes to obtain an enzymolysis solution.

[0022] In some embodiments of the present invention, the protease is alkaline protease, trypsin, pepsin, neutral protease and papain; preferably alkaline protease, trypsin, neutral protease and papain; more preferably, the enzyme activity is 200000-300000 U / ml.

[0023] In some embodiments of the present invention, the added amount of the protease is 1%-5% of the protein amount in the quinoa protein solution.

[0024] In some embodiments of the present invention, in step S4, in the ultrafiltration step, the enzymatic hydrolysate with a molecular weight cutoff of ≤5 kDa is retained.

[0025] In some more preferred embodiments of the present invention, in step S4, the enzymatic hydrolysate with a molecular weight cutoff of ≤2 kDa is retained.

[0026] Furthermore, in step S5, the amount of water-soluble iron salt added is 20%-60% of the amount of quinoa protein peptide.

[0027] In some embodiments of the present invention, in step S5, the concentration of quinoa protein peptide is 2-10 mg / ml.

[0028] In some embodiments of the present invention, the water-soluble iron salt is selected from one or a combination of ferrous chloride, ferrous sulfate, ferrous fumarate, and ferrous lactate.

[0029] In some embodiments of the present invention, in step S6, in the alcohol precipitation step, an excess volume of anhydrous ethanol is added, the mixture is allowed to stand for 2-3 hours, and then centrifuged and the precipitate is collected and freeze-dried.

[0030] In some preferred embodiments, in step S6, the amount of anhydrous ethanol used is 4-6 times that of the chelating solution.

[0031] In some preferred embodiments, the method for preparing quinoa protein peptide-iron chelate comprises the following steps:

[0032] (1) Extraction of quinoa protein: Take quinoa powder and add n-hexane and stir overnight. Change the liquid two to three times to remove fat. After drying, add appropriate amount of water, adjust the pH value to 9-10, stir evenly for a certain period of time, filter the precipitate to obtain protein extract. Adjust the pH to the isoelectric point, and centrifuge to obtain protein precipitate. Wash the precipitate with water multiple times to improve the purity of the protein. Freeze-dry for later use.

[0033] (2) Inoculation and fermentation: The fermentation conditions were optimized through single factor experiments, including substrate concentration, inoculation concentration, fermentation time, etc. The optimal conditions were a water to quinoa protein ratio of 1:20-1:40 and an inoculation concentration of 10 7 -10 8 CFU / mL, fermentation time was 24 hours.

[0034] (3) Enzymatic hydrolysis: After fermentation, the fermentation broth can be enzymatically hydrolyzed to further hydrolyze the peptide chain and produce smaller peptide segments, which usually have better metal chelating ability. The pH value of the quinoa protein solution is adjusted to 8.0-9.0, and protease is added. After enzymatic hydrolysis at 50℃-55℃ for 3-4h, the enzymatic hydrolyzate is heated to 95℃ for 10min to inactivate the enzyme, and the enzymatic hydrolyzate is obtained.

[0035] (4) Ultrafiltration: The enzymatic hydrolyzate obtained in step (3) is ultrafiltered through a ceramic membrane device with a molecular weight cutoff of 2000Da and 5000Da, and the permeate is collected to obtain a quinoa protein peptide solution;

[0036] (5) Chelating reaction: adding a water-soluble iron salt to the quinoa protein peptide solution obtained in step (4), wherein the amount of the water-soluble iron salt added is 20%-60% of the amount of the quinoa protein peptide (based on protein content), adjusting the pH value of the solution to 6.0-8.0, stirring the chelating reaction at 30° C.-50° C. for 1-2 hours, and obtaining a quinoa protein peptide-iron chelating reaction solution;

[0037] (6) Freeze drying: add an excess volume of anhydrous ethanol to the quinoa protein peptide-iron chelation reaction solution obtained in step (5), let stand for 2-3 hours, and centrifuge at 10000 r / min for 15 minutes; collect the precipitate and freeze-dry it for later use.

[0038] The second aspect of the present invention is to provide quinoa protein peptide-iron chelate prepared by the preparation method described above. The prepared iron chelate peptides of different molecular weights will be screened by using in vitro digestion stability and chelation rate as indicators.

[0039] Furthermore, the amino acid sequence of the peptide-iron chelate is: Arg-Leu-Val-Leu-Pro-Gly-Glu-Leu-Ala-Lys-His; Ala-Ile-Asp-Arg-Pro-Glu-Gly-Phe.

[0040] By implementing the above technical solution, the present invention has the following beneficial effects:

[0041] (1) The present invention comprehensively applies modern bioengineering technologies such as microbial fermentation, bioenzymatic hydrolysis, ultrafiltration, molecular chelation and freeze drying to prepare a quinoa protein peptide-iron chelate; the preparation method has a simple process flow and high production efficiency, and is suitable for industrial-scale production;

[0042] (2) The quinoa protein peptide-iron chelate prepared by the present invention uses nutrient-rich quinoa protein as a peptide source and an inorganic iron salt as an iron source, and the peptide-iron chelate has a small molecule peptide content of more than 80%, a chelated iron ion content of more than 10%, and a chelation rate of more than 98%;

[0043] (3) The quinoa protein peptide-iron chelate prepared by the present invention has high stability during gastric digestion and is less irritating to the stomach; and releases iron during intestinal digestion, which is easier to be digested and absorbed by the human body in the intestine, and can achieve a better iron supplementation effect;

[0044] (4) The quinoa protein peptide-iron chelate prepared by the present invention is safe, has no toxic side effects, promotes iron absorption, and can form a stable polypeptide-iron chelate. Due to its unique chelation mechanism and transport mechanism, it is easily absorbed by the human body. In addition, the quinoa protein peptide does not contain gluten, will not cause allergies, and is applicable to a wider range of people. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The accompanying drawings are used to provide a further understanding of the present application and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the present application and do not constitute a limitation of the present application. In the accompanying drawings:

[0046] Figure 1 The in vitro digestion stability of the peptide iron chelates prepared in Examples 1-5 (Examples 1-5 correspond to ae, respectively);

[0047] Figure 2 The peptide iron chelation rates of peptides of different molecular weights prepared in Examples 1-5;

[0048] Figure 3 Molecular docking diagram;

[0049] Figure 4 Comparison of the chelation rates of the peptide iron chelates obtained in Example 1 and Comparative Example 1;

[0050] Figure 5 Infrared spectrum analysis of QPP and its iron chelate (QPP-Fe) prepared in Example 1;

[0051] Figure 6 Ultraviolet spectrum analysis of QPP and its iron chelate (QPP-Fe) prepared in Example 1. DETAILED DESCRIPTION

[0052] In order to make the above-mentioned purpose, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the embodiments of the specification. These embodiments are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions or conditions recommended by the manufacturer.

[0053] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. The present invention is not limited to the specific embodiments disclosed below.

[0054] Test characterization methods:

[0055] Peptide molecular weight detection: High performance gel filtration chromatography (HPLC) was used for determination, with proteins of known molecular weight as reference.

[0056] Iron content detection: Atomic absorption spectrophotometer was used to determine the total iron content and the chelated iron content in quinoa protein peptide iron chelate, and the chelation rate of soybean protein peptide iron chelate was calculated based on the results.

[0057] Determination of peptide iron chelation rate: Referring to the method of (Zhao Jing. Preparation and property study of pig hemoglobin iron chelating peptide [D]. Jilin University, 2016.), the chelation rate of peptide and ferrous ion was determined.

[0058]

[0059] Mix 50 μL QPP solution (1%, w / v) with 130 μL sodium acetate buffer solution (1M, pH=6), add 20 μL FeCl2·4H2O (2mM), and add to a 96-well plate. Incubate at 37°C for 30 min. Add 10 μL (5mM) phenanthroline and react for 10 min in the absence of light. Replace QPP solution with water as a total iron control, and replace FeCl2·4H2O with water as a blank control. Measure the light absorption value of the colored complex at 562 nm.

[0060] Simulated gastric fluid: Accurately weigh 0.2 g sodium chloride and 3 g pepsin (enzyme activity 15000 U / g), measure 80 mL of water, put them in a 100 mL beaker, stir the mixture to make it fully dissolved, then adjust the pH of the mixture to 2.0 with 1 mol / L HCl and make up to volume in a 100 mL volumetric flask. Prepare 5 mg / mL peptide iron chelate (QPP-Fe) and 1 mg / mL FeSO4 aqueous solution respectively, pipette 10 mL of each solution into a conical flask, adjust the pH to 2.0 with 1 mol / L hydrochloric acid, put the conical flask into a 37°C constant temperature shaking incubator for preheating for 5 min, add 10 mL of simulated gastric juice and mix well, react in a 37°C constant temperature shaking incubator at 150 rpm for 0, 10, 30, 60, 120, and 180 min, take out and inactivate the enzyme in a 95°C water bath for 10 min, let it stand and cool, centrifuge at 8000 r / min for 10 min, collect the supernatant, and determine the iron concentration in the sample solution by phenanthroline colorimetry.

[0061] Simulated intestinal fluid: 5.40g sodium chloride, 0.65g potassium chloride and 0.33g calcium chloride dihydrate were dissolved in 1L distilled water to prepare intestinal electrolyte solution, 7% trypsin-PBS solution and 4% bile salt-PBS solution. 50mL intestinal electrolyte, 50mL trypsin-PBS solution, 100mL bile salt-PBS solution, 59.50mg pancreatic lipase and 13.00mg trypsin were added to the remaining gastric digestive fluid, the pH was adjusted to 7 at 37℃ with 0.10mol / L sodium hydroxide solution, and incubated at 80r / min for 2h. Samples were collected at 20, 40, 120, 140, 160, 180 and 240min respectively. The enzyme was inactivated in a 95℃ water bath for 10 minutes, centrifuged at 8000r / min and centrifuged at 4℃ for 15 minutes. The supernatant was taken and freeze-dried as above to obtain the gastrointestinal digestion product. The iron concentration in the sample solution was determined by phenanthroline colorimetry.

[0062] UV analysis: UV spectroscopy was used to scan the quinoa protein peptide component (QPP) and its iron chelate (QPP-Fe) to qualitatively observe the changes in the sample's UV absorption spectrum. A certain amount of QPP and QPP-Fe were weighed, respectively, dissolved in distilled water to prepare a sample solution with a concentration of 0.2 mg / mL, and the UV spectrum was scanned in the wavelength range of 200-800 nm.

[0063] Infrared analysis: QPP and QPP-Fe were analyzed by infrared spectroscopy using the KBr tablet method. KBr was placed in an infrared drying oven and dried to constant weight. 1 mg of QPP and QPP-Fe samples were accurately weighed and ground with 100 mg of KBr powder. After grinding evenly, they were pressed into thin sheets using a tablet press. The samples were scanned and measured using a Fourier transform infrared spectrometer with a scanning range of 4000 to 400 cm -1 , scanning times 32 times, resolution 2cm -1 .

[0064] Fermentation strain screening

[0065] Lactobacillus, Bacillus, Morganella, and Citrobacter strains were selected and cultured in Luria-Bertani (LB) or De Man, Rogosa, Sharpe (MRS) medium for 24 hours at 37°C and a shaking speed of 200 rpm. After culture, the bacteria were collected and their concentration was adjusted to 10 7 -10 8 CFU / mL.

[0066] Fermentation experiment: 1 mL of bacterial suspension was inoculated into a fermentation medium containing 5 g of quinoa protein and fermented for 24 hours.

[0067] After fermentation, the fermentation broth was treated with enzymatic hydrolysis, and the peptide content was determined by BCA kit, and the iron chelation rate was determined by Ferrozine method. The iron chelation rate was used as the main screening index, and the peptide content was used as the secondary index.

[0068] Among them, lactic acid bacteria (Lactobacillus) have a strong ability to ferment proteins and enhance nutritional value through metabolites. Therefore, through the comparison of the final experimental results, lactic acid bacteria showed the highest iron chelation rate (98.94%) after fermentation, and the peptide content was also high, so it was selected as the best fermentation strain. We finally selected Lactobacillus as the best fermentation strain.

[0069] Example 1

[0070] (1) Extraction of quinoa protein: Weigh 10 kg of quinoa powder, add n-hexane at a ratio of 1:4 (w / v), stir overnight, and replace the liquid three times to remove fat. After drying, add 1:12 (w / v) water, adjust the pH value to 9, stir for 3 hours, filter the precipitate to obtain a protein extract. Adjust the pH to the isoelectric point, and centrifuge to obtain a protein precipitate. Wash the precipitate three times with water to improve the protein purity. Freeze-dry for later use.

[0071] (2) Preparation of quinoa protein solution: Weigh 1 kg of quinoa protein powder (86%) and add 20 kg of water and stir evenly to prepare a quinoa protein solution with a protein concentration of 5% (w / w).

[0072] (3) Inoculation and fermentation: 200 ml of 10 7 -10 8 The CFU / mL lactic acid bacteria strain was inoculated into the protein solution and fermented at 37°C for 24 hours.

[0073] (4) Enzymatic hydrolysis: The pH value of the quinoa protein fermentation liquid was adjusted to 8.0, and alkaline protease was added. After enzymatic hydrolysis at 55°C for 3 hours, the enzymatic hydrolyzate was heated to 95°C for 10 minutes to inactivate the enzyme, thereby obtaining an enzymatic hydrolyzate. The supernatant was collected after centrifugation at 10,000 r / min for 10 minutes.

[0074] (5) Ultrafiltration: ultrafiltration of the supernatant obtained in step (4) using a ceramic membrane device with a molecular weight cutoff of 2000Da and 5000Da, collecting the permeate to obtain a quinoa protein peptide solution;

[0075] (6) Chelating reaction: adding a water-soluble iron salt to the quinoa protein peptide solution obtained in step (5), wherein the amount of the water-soluble iron salt added is 20% of the amount of the quinoa protein peptide (based on protein content), adjusting the pH value of the solution to 6.0, and stirring the chelating reaction at 37° C. for 1 hour to obtain a quinoa protein peptide-iron chelating reaction solution;

[0076] (7) Freeze drying: add an excess volume of anhydrous ethanol to the quinoa protein peptide-iron chelation reaction solution obtained in step (6), let it stand for 2 hours, and centrifuge at 10,000 r / min for 15 minutes; collect the precipitate and freeze-dry it.

[0077] Example 2

[0078] The preparation method of Example 2 is the same as that of Example 1, except that the pH of the enzymatic hydrolysis solution is adjusted to 8.5, and trypsin is added for enzymatic hydrolysis.

[0079] Example 3

[0080] The preparation method of Example 3 is the same as that of Example 1, except that the pH of the enzymatic hydrolysis solution is adjusted to 7, and neutral protease is added for enzymatic hydrolysis.

[0081] Example 4

[0082] The preparation method of Example 4 is the same as that of Example 1, except that the pH of the enzymatic hydrolysis solution is adjusted to 2.5, and pepsin is added for enzymatic hydrolysis.

[0083] Example 5

[0084] The preparation method of Example 5 is the same as that of Example 1, except that the pH of the enzymatic hydrolysis solution is adjusted to 8, and papain is added for enzymatic hydrolysis.

[0085] Example 6

[0086] The preparation method is basically the same as that in Example 1, except that the enzymatic hydrolysis step controls the enzymatic hydrolysis time to 0.5, 1, 2, 3, 4, and 5 h, each group of enzymatic hydrolysis reactions is tested in parallel three times, and the ferrous chelating ability and hydrolysis degree determination experiments are carried out in parallel three times.

[0087] Example 7

[0088] The preparation method is basically the same as that in Example 1, except that the enzymatic hydrolysis temperatures are (37°C, 55°C, 55°C, 55°C, 37°C), respectively, and each group of enzymatic hydrolysis reactions is tested in parallel three times, and the ferrous chelating ability and hydrolysis degree determination experiments are carried out in parallel three times.

[0089] Example 8 Different substrate concentrations

[0090] The preparation method is basically the same as that in Example 1, except that the prepared quinoa protein peptide at different concentrations (0.1, 0.5, 1, 2, 4, 8, 10 mg / ml) was reacted with ferrous iron (2:1) in a shaking water bath at 37°C for 2 hours, and the ferrous iron chelation rate was measured. The results are shown in Table 1:

[0091] Table 1

[0092]

[0093] The results of Examples 1-8 show that the best effect of alkaline protease hydrolysis of quinoa protein is achieved after lactic acid bacteria fermentation. The optimal process conditions are pH 8.0, enzyme addition amount 50:1, enzymatic hydrolysis temperature 55°C, substrate mass concentration 5 mg / ml, and enzymatic hydrolysis time 3 h. Under the optimal enzymatic hydrolysis conditions, the ferrous chelating capacity is (78.6±0.02) mg / g.

[0094] Comparative Example 1 does not have a fermentation step

[0095] The experimental method and steps are basically the same as those in Example 1, except that there is no fermentation step.

[0096] The quinoa protein peptide prepared in Example 1 and Comparative Example 1 was reacted with ferrous iron in a shaking water bath at 37° C. for 2 hours, and the ferrous iron chelation rate was measured. Figure 4 As shown, it can be seen that the quinoa protein peptide obtained by fermentation and then enzymatic hydrolysis has a higher chelation rate with iron.

[0097] Experimental Example 1: Ultraviolet and infrared spectroscopic analysis of QPP and QPP-Fe prepared in Example 1:

[0098] UV-visible spectroscopy is an effective tool for analyzing the formation of QPP-Fe chelates. Due to the chelation of iron ions with peptides, the original absorption peak shifts or disappears. Figure 6 As shown, the QPP prepared in Example 1 has maximum absorption peaks at 210nm and 265nm. 2+ After chelation to form QPP-Fe chelate, these two absorption peaks disappeared, and the absorption intensity of the chelate was higher than that of QPP. This phenomenon may be due to the change in the chiral spatial configuration of the chromophores on the peptide (CO and -COOH) and the peptide backbone (-OH and -NH2) during the binding process of ferrous ions. This is because the oxygen atom on the carbonyl group participates in the Fe 2+ The complexation of Fe 2+ The addition of caused charge transfer transition and coordination field transition, resulting in a change in the charge of the carbonyl group. This shows that the chelate is two different substances from the unchelated state. This change promotes the charge transfer between tyrosine, tryptophan and phenylalanine. These adaptabilities indicate that QPP can bind to iron ions to form a QPP-Fe chelate.

[0099] Figure 5 The infrared spectra of QPP and QPP-Fe show that the changes in the absorption peaks indicate that a coordination reaction has occurred between the ferrous ions and the QPP functional groups. -1 wavelength, FTIR spectra of QPP before and after chelation with ferrous ions Figure 5 As shown. QPP at 3387.71cm -1 However, when QPP and Fe 2+ When chelated, the characteristic absorption peak moves to 3406.79 cm -1 , indicating that amino acids are complexed with iron ions through the NH band. QPP is at 1650.07 cm -1 The absorption peak at corresponds to amide I (1700-1600 cm -1 ), and after combining with iron, due to the stretching vibration of CO, the absorption peak of QPP-Fe chelate changes from 1650.07 cm -1 Move to 1653.35cm -1The results show that the vibration intensity of CO increases in the QPP-Fe chelate, indicating that the intramolecular hydrogen bonding force decreases and the amount of CO providing iron ion coordination increases. The obvious peak of the -coo- group associated with the QPP-Fe chelate increases from 1455.76 cm -1 Move to 1322.26cm -1 . This result originates from the binding of carboxyl groups to ferrous iron. This indicates that the chelating site of iron is also related to the C-terminal carboxylic acid and glutamic acid and aspartic acid residues. It can be seen that the -NH2 and -COOH functional groups of QPP are the main sites for iron binding. The nitrogen and oxygen in the peptide bond each have a pair of lone electrons. These electrons interact with the iron ion to form a chelate with a ring structure.

[0100] Experimental Example 2: Chelating ability of quinoa protein peptides with different molecular weights

[0101] The quinoa protein peptide (QPP) obtained in Example 1-5 was purified by ultrafiltration, and QPP was prepared into a 10 mg / mL solution. Subsequently, at room temperature and a pressure of 0.5 MPa, the solution was passed through a 5 kDa and 2 kDa ceramic membrane in sequence to obtain three components of >5 kDa, 5 kDa to 2 kDa, and <2 kDa. At the same time, the measured ferrous chelation rate is shown in Table 2.

[0102] Table 2 Determination of protein content and ferrous chelation capacity of ultrafiltration components of Examples 1-5 (chelation rate %)

[0103]

[0104] like Figure 2 As shown in Table 1, among the three components obtained by ultrafiltration, the component protein peptide <2kDa has the highest ferrous chelation activity, so this component is retained. The chelation rate of ferrous ions with peptides less than 2000Da can reach up to 98% (78.6mg / g), which is higher than the ferrous chelation rate of peptides in other intervals.

[0105] Experimental Example 3: Analysis of Molecular Docking Results

[0106] Take an appropriate amount of sample and desalt it through a C18 desalting column. The sample was analyzed by LC-MS / MS equipped with an online nanospray ion source. The whole system was an Orbitrap Fusion Lumos mass spectrometer (ThermoFisher Scientific, MA, USA) connected in series with an EASY-nanoLC 1200. A total of 5 μL of sample was loaded (C18 column: 20 cm × 75 μm id, 1.9 μm particle size), and the sample was separated by a gradient of 60 min. The column flow rate was controlled at 300 nL / min, the column temperature was 40 °C, the electrospray voltage was 2 kV, and the gradient started from 4% of phase B, and increased to 50% in 53 min 40 s with a nonlinear gradient, and increased to 95% within 40 s, and maintained for 5 min 40 s.

[0107] Select amino acids that are likely to bind to ferrous ions from the identified peptide sequences. For example, peptides rich in amino acids such as aspartic acid (Asp), glutamic acid (Glu), and histidine (His) are usually more likely to bind to metal ions. Among them, AIDRPEGF contains fewer amino acids that bind to ferrous ions through four bonds; and forms an encapsulation state for ferrous ions. RLVLPGELAKH has a higher binding energy for ferrous ions (-7.18 kJ / mol). AIDRPEGF and RLVLPGELAKH were selected for molecular docking simulation with ferrous ions. Molecular docking software (such as AutoDock or MOE) was used to simulate the binding process of peptides with ferrous ions, analyze their binding mode and binding energy, and predict binding ability.

[0108] Fe 2+ Molecular docking was performed with AIDRPEGF and RLVLPGELAKH targets. The results are as follows Figure 3 As shown in the figure, the molecular docking results show that the metal ions and the target peptides have a good binding effect and a good match. The complex formed by the metal ions and the peptides after docking was visualized using Pymol2.1 software to obtain the binding mode of the metal ions and the peptides. According to the binding mode, the amino acid residues that bind to the metal ions and the peptide pockets can be clearly seen. For example: Fe 2+ The metal ions form electrostatic interactions with the amino acids GLU-7 and HIS-11 at the RLVLPGELAKH site, which stabilizes the Fe 2+ Fe plays an important role. 2 + The metal ions form coordination with the negatively charged ALA-1, ARG-4, PRO-5, and GLY-7 of the AIDRPEGF polypeptide, which is beneficial to stabilizing the metal ions. The molecular docking results are shown in Table 3.

[0109] Table 3

[0110] Peptide chain Ferrous ion Docking force (kJ / mol) Combination Type AIDRPEGF <![CDATA[Fe 2+ ]]> -6.23 Electrostatic interactions RLVLPGELAKH <![CDATA[Fe 2+ ]]> -7.18 Electrostatic interactions

[0111] Arg-Leu-Val-Leu-Pro-Gly-Glu-Leu-Ala-Lys-His(RLVLPGELAKH);

[0112] Ala-Ile-Asp-Arg-Pro-Glu-Gly-Phe(AIDRPEGF);

[0113] Experimental Example 4: In vitro gastrointestinal digestion of quinoa protein peptide iron chelate:

[0114] The results of simulating in vitro gastrointestinal digestion of the peptide iron chelates prepared in Examples 1-5 are as follows: Figure 1 As shown, the free amount of ferrous ions in the stomach of quinoa protein peptide iron chelate is about 20%, which is significantly lower than the iron ion release of FeSO4. Therefore, the quinoa protein peptide iron chelate is more stable during gastric digestion and less irritating to the stomach. In the digestion process of the intestine, the iron release of quinoa protein peptide iron chelate is significantly higher than that of ferrous sulfate. This is because the ferrous ions released by ferrous sulfate in the stomach during intestinal digestion are oxidized into trivalent iron to form non-absorbable precipitation. Quinoa protein peptide iron chelate can better release ferrous ions during intestinal digestion, thereby improving intestinal absorption. Therefore, the quinoa protein peptide iron chelate prepared by the present invention has better stability in the stomach and less irritating to the stomach than ferrous sulfate; it is easier to be digested and absorbed by the human body in the intestine, and can play an effective iron supplement effect.

[0115] In summary, the present invention comprehensively applies modern bioengineering technologies such as microbial fermentation, bio-enzymolysis, ultrafiltration, molecular chelation and freeze-drying to prepare a quinoa protein peptide-iron chelate; the preparation method has a simple process flow and high production efficiency, and is suitable for industrial-scale production; the nutrient-rich quinoa protein is used as the peptide source, and the inorganic iron salt is used as the iron source, and the peptide-iron chelate is prepared, and the content of small molecule peptides (relative molecular weight between 180 and 1000 Da) is as high as 80% or more, the content of chelated iron ions is more than 10%, and the chelation rate reaches more than 98%; the prepared quinoa protein peptide-iron chelate has high stability during gastric digestion and is less irritating to the stomach; it is easier to be digested and absorbed by the human body in the intestine, and has a better iron supplement effect; the prepared quinoa protein peptide-iron chelate is safe and has no toxic side effects, the quinoa protein peptide does not contain gluten, will not cause allergies, and is applicable to a wider range of people.

[0116] The above are only several embodiments of the present invention and are not intended to limit the present invention in any form. Although the present invention is disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, using the above disclosed technical content to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A method for preparing quinoa protein peptide-iron chelate, characterized in that: The following steps are involved: S1. Extraction of quinoa protein: taking quinoa powder, adding solvent to extract and defatting, solid-liquid separation, drying, adding water, adjusting the pH value to 9-10, stirring evenly, filtering the precipitate, and obtaining a protein extract; adjusting the pH of the protein extract to the isoelectric point, centrifuging, washing, and drying to obtain quinoa protein; S2. Inoculation and fermentation: dissolving the quinoa protein obtained in step S1 in water to prepare a quinoa protein solution, and inoculating lactic acid bacteria for fermentation; S3 enzymatic hydrolysis: taking the fermentation broth obtained in step S2, adding protease, enzymatic hydrolysis, to obtain an enzymatic solution; S4. Ultrafiltration: Take the hydrolyzate obtained in step S3, and use a ceramic membrane of 2000Da and 5000Da to retain the hydrolyzate with a molecular weight cutoff of 0-2000Da, 2000Da-5000Da and>5000Da to obtain a quinoa protein peptide solution; S5. Chelation reaction: Add a water-soluble iron salt to the quinoa protein peptide solution obtained in step S4, adjust the pH to 6.0-8.0, and stir the chelation reaction at 30°C-50°C to obtain a quinoa protein peptide-iron chelation reaction solution; S6. Take the reaction solution obtained in step S5, collect the precipitate after alcohol precipitation to obtain quinoa protein peptide-iron chelate.

2. The method for preparing quinoa protein peptide-iron chelate according to claim 1, characterized in that: In step S1, the extraction solvent is selected from n-hexane and deionized water; The ratio of quinoa powder to solvent (w / v) is 1:4-1:

12.

3. The method for preparing quinoa protein peptide-iron chelate according to claim 1, characterized in that: In step S2, the lactic acid bacteria are selected from Lactobacillus plantarum, Lactobacillus bulgaricus and Streptococcus thermophilus; And / or, in step S2, the fermentation conditions are: the ratio of water to quinoa protein is 1:20-1:40, the inoculation concentration is 10 7 -10 8 CFU / mL, fermentation time is 24-48 hours.

4. The method for preparing quinoa protein peptide-iron chelate according to claim 1, characterized in that: In step S3, the pH value of the quinoa protein solution is adjusted to 8.0-9.0, and protease is added. After enzymolysis at 50° C.-55° C. for 3-4 hours, the temperature is raised to inactivate the enzyme to obtain an enzymolysis solution.

5. The method for preparing quinoa protein peptide-iron chelate according to claim 1, characterized in that: The proteases are alkaline protease, trypsin, neutral protease and papain; in step S3, the enzyme activity of the protease is 200000-300000 U / ml.

6. The method for preparing quinoa protein peptide-iron chelate according to claim 1, characterized in that: In step S3, the added amount of the protease is 1%-5% of the protein amount in the quinoa protein solution.

7. The method for preparing quinoa protein peptide-iron chelate according to claim 1, characterized in that: In step S4, in the ultrafiltration step, the enzymatic hydrolysate with a molecular weight cutoff of ≤5 kDa is retained.

8. The method for preparing quinoa protein peptide-iron chelate according to claim 1, characterized in that: In step S4, the enzymatic hydrolysate with a molecular weight cutoff of ≤2 kDa is retained.

9. The method for preparing quinoa protein peptide-iron chelate according to claim 1, characterized in that: In step S5, the amount of water-soluble iron salt added is 20%-60% of the amount of quinoa protein peptide; And / or, the water-soluble iron salt is selected from one or a combination of ferrous chloride, ferrous sulfate, ferrous fumarate, and ferrous lactate; And / or, in step S5, the concentration of quinoa protein peptide is 2-10 mg / ml.

10. The method for preparing quinoa protein peptide-iron chelate according to claim 1, characterized in that: In step S6, in the alcohol precipitation step, an excess volume of anhydrous ethanol is added, the mixture is allowed to stand for 2-3 hours, and then centrifuged, the precipitate is collected and freeze-dried; And / or, in step S6, the amount of anhydrous ethanol used is 4-6 times that of the chelating solution.

11. Quinoa protein peptide-iron chelate prepared by the method according to any one of claims 1 to 10.

12. The quinoa protein peptide-iron chelate according to claim 11, characterized in that The amino acid sequence of the peptide-iron chelate is: Arg-Leu-Val-Leu-Pro-Gly-Glu-Leu-Ala-Lys-His; or Ala-Ile-Asp-Arg-Pro-Glu-Gly-Phe.

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