Iron-chelating Peptides Research Articles

A novel antioxidant iron-chelating peptide from yak skin: analysis of the chelating mechanism and digestion stability in vitro.

The global prevalence of iron deficiency has posed significant public health risks. Animal-derived collagen peptides have been recognized for their potent metal ion-chelating capabilities, which can greatly enhance the bioavailability of iron. Yak skins, typically discarded during production and processing, serve as a valuable resource. Based on yak skin collagen peptide (YSP), we have developed a novel iron-chelating peptide: yak skin collagen iron-chelating peptide (YSP-Fe). The maximum level of iron chelation of YSP-Fe achieved was 42.72 ± 0.65 mg g-1. Structural analysis indicated that YSP-Fe was primarily formed from amino, carboxyl and carbonyl groups combined with ferrous ions. Through examination of the amino acid composition, molecular docking and peptide sequence identification, it was determined that Gly, Asp and Arg played crucial roles in the chelation of ferrous ions by YSP. Furthermore, YSP-Fe was more stable in simulated gastrointestinal digestion compared to FeSO4. YSP-Fe demonstrated dual benefits of iron supplementation and antioxidant effects. These significant findings provide a foundation for the development of novel iron supplements and the effective utilization of yak skin as a valuable resource. © 2024 Society of Chemical Industry.

Excellent iron-chelating capacity of Yesso scallop (Patinopecten yessoensis) skirt hydrolysate fermented by Bacillus subtilis M17-b7

Peptide ferrous chelate is a new type of biological iron, which has the advantages of high absorption rate, safety and no digestive tract irritation. In this study, microbial fermentation was used for the first time to prepare iron-chelating peptides. Forty-eight strains were applied to ferment scallop skirts. A dominating strain was eliminated by using the iron binding capacity of the scallop skirt hydrolysate as the screening index. The scallop skirt hydrolysate fermented by Bacillus subtilis M17-b7 showed excellent iron-chelating capacity (96.20% ± 0.03%). Meanwhile, the structure, morphology and bioactivity of fermented scallop skirt hydrolysate (FSH) and fermented scallop skirt hydrolysate-iron (FSH–Fe) were measured. Based on the amino acid composition and spectral analysis, it was confirmed that the significant amino acids contains carboxyl, carbonyl, and amino groups. The result of scanning electron microscope indicated that the surface of FSH became porous and the particles were bigger after chelating with ferrous. Moreover, the peptide-ferrous chelate was more stable in salt solutions and simulated gastric juice compared with ferrous sulfate. It is also noteworthy that the preparation of ferrous chelate peptides by fermentation is cost-effective compared with enzymatic hydrolysis. Therefore, this study provides theoretical support for the use of fermentation as a promising tool to produce peptide-ferrous chelate.

Novel iron-chelating peptide from egg yolk: Preparation, characterization, and iron transportation.

In this work, an egg yolk protein hydrolysate (EYPH) with a high iron-chelating ability (87.32%) was prepared. The fractionation using 60% (v/v) ethanol concentration (E3 fraction) led to the efficiently accumulating the iron-chelating peptides in EYPH. The characterization results showed that iron mainly chelated with carboxyl, amino and phosphate groups of peptides. From E3 fraction, six iron-chelating peptides with MW ranging from 1372.36 to 2937.04Da were identified and a hypothesized molecular model of DDSSSpSpSpSpSpSVLSK-Fe was simulated. In vitro stability determination showed that E3-Fe chelate owned a good heat, alkalinity and digestion tolerance, but a relatively bad acid tolerance. Finally, iron transport analysis showed that iron in the E3-Fe would be absorbed in caco-2 cell membrane more effectively than that of iron salts, indicating that it was possible to apply the E3-Fe complex as iron supplements.

Identification of high iron–chelating peptides with unusual antioxidant effect from sea cucumbers and the possible binding mode

Iron deficiency anemia (IDA) is a common nutritional disease affecting 2 billion people. To develop a new iron-fortified food, we designed a novel type of iron-chelating peptide [Sea cucumbers peptides (SCP)–Fe] from sea cucumbers. SCP can chelate ferrous ions. The neutral protease hydrolysate have the highest iron chelating activity (117.17 ± 2.62 mg/g). Single factors including pH, material ratio, and molecular weight, had a significant effect on the iron chelating activity. The characterization of the SCP–Fe chelate revealed a loose and blocky structure with increased particle size. The amino acid composition, peptide identification and molecular docking indicated that Asp, Glu, Gly and Pro played an important role in binding to ferrous ions. After chelation, SCP–Fe chelate had dual nutrition effects of stronger radical scavenging ability and potential high-efficiency iron supplementation ability. These results might provide insights into the methods for developing functional foods such as iron-fortified seafood.

Optimization of ultrasound assisted extraction of abalone viscera protein and its effect on the iron-chelating activity

This study aims to investigate effects of ultrasound assisted extraction on the abalone viscera protein extraction rate and iron-chelating activity of peptides. The optimal conditions for ultrasound assisted extraction by response surface methodology was at sodium hydroxide concentration 14 g/kg, ultrasonic power 428 W and extraction time 52 min. Under the optimal conditions, protein extraction rate was 64.89%, compared with alkaline extraction of 55.67%. The iron-chelating activity of peptides affected by ultrasound technology was further evaluated by iron-chelating rate, FTIR spectroscopy and LC-HRMS/MS. Alcalase was the suitable enzyme for the preparation of iron-chelating peptides from two abalone viscera proteins, showing no significant difference between their iron-chelating rate of 16.24% (ultrasound assisted extraction) and 16.60% (alkaline extraction). Iron binding sites from the two hydrolysates include amino and carboxylate terminal groups and peptide bond of the peptide backbone as well as amino, imine and carboxylate from side chain groups. Moreover, 24 iron-chelating peptides were identified from hydrolysate (alcalase, ultrasound assisted extraction), which were different from the 27 iron-chelating peptides from hydrolysate (alcalase, alkaline extraction). This study suggests the application of ultrasound technology in the generation of abalone viscera-derived iron-chelating peptides which have the ability to combat iron deficiency.

The Preparation, Antioxidant Activity Evaluation, and Iron-Deficient Anemic Improvement of Oat (Avena sativa L.) Peptides-Ferrous Chelate.

Iron-chelating peptides have been widely considered as one of the best iron supplements to alleviate the iron deficiency. In this study, a novel oat peptides–ferrous (OP-Fe2+) chelate was prepared from antioxidant oat peptides obtained in the laboratory of the authors. The optimal preparation condition was obtained through the single-factor and response surface methodology, and the chelating rate could reach up to 62.6%. After chelation, the OP-Fe2+ chelate exhibited a significantly higher 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity than oat peptides. It was discovered that the hemoglobin concentration and the number of red blood cell levels in OP-Fe2+-treated iron-deficient anemic (IDA) rats were significantly higher than untreated IDA rats. The OP-Fe2+ chelate could also improve the hypertrophy of the spleen, serum iron (SI), total iron and binding capacity, and serum ferritin levels in the IDA rats. In addition, the OP-Fe2+ treatment significantly increased the antioxidant activities of super oxidase and glutathione in the liver homogenate of the IDA rats. Therefore, the OP-Fe2+ chelate is an effective type of iron supplement for IDA rats, which could be a promising source with anti-anemia and antioxidant activity.

Preparation, purification and identification of iron-chelating peptides derived from tilapia (Oreochromis niloticus) skin collagen and characterization of the peptide-iron complexes

Tilapia skin collagen was hydrolyzed by five proteases (trypsin, pepsin, neutral protease, alkaline protease and protamex). The results showed that the tilapia skin collagen hydrolysate (TSCH) obtained by 2 h hydrolysis with trypsin exhibited the highest iron chelating rate. The TSCH was then separated by immobilized metal affinity chromatography (IMAC-Fe2+) and obtained the tilapia skin collagen iron-chelating peptides (TSCICP). The iron chelating sites of TSCICP were corresponding to carboxylic groups of Asp/Glu and guanidine nitrogen of Arg/Lys. After chelated with iron ions, TSCICP was folded and aggregated to form spherical particles with increased particle size. The TSCICP-iron complexes could maintain high iron solubility at various pH and in simulated gastrointestinal digestion. The iron bio-accessibility of TSCICP-iron complexes was high than that of ferrous glycinate and ferrous sulfate. Finally, TSCICP was purified by RP-HPLC and identified by LC-MS/MS. Four iron-chelating peptides were identified as GPAGPAGEK (782.39 Da), DGPSGPKGDR (984.46 Da), GLPGPSGEEGKR (1198.59 Da) and DGPSGPKGDRGETGL (1441.68 Da). These results indicating that the iron-chelating peptides derived from tilapia skin collagen could be used as potential dietary iron supplement.

Preparation and characterization of iron-chelating peptides from whey protein: An alternative approach for chemical iron fortification

Iron fortification of staple food is a strategy utilized worldwide to address the concern of dietary iron deficiency. However, traditional salt-based fortification methods have limitations with gastrointestinal stability and bioavailability. Iron chelating peptides from easily available and scalable proteins such as whey protein have been proposed as promising candidates to circumvent the above mentioned limitations by enhancing iron absorption and bioavailability. In this study, we report methods to produce whey protein derived iron-chelating peptides and describe their physicochemical characteristics. Peptides derived from whey proteins prepared by ultrafiltration of whey followed by hydrolysation were iron chelated to produce peptide-iron complexes. These complexes had a size of 422.9 ± 3.41 nm, chelated iron content of 36.42 µg/ mg protein, and a low zeta potential (−10.80 mV) compared to whey peptides. Spectra analysis using ultraviolet–visible absorption and Fourier transform infrared spectroscopy showed structural transformation indicating iron chelation. Mass spectrometric analysis using LC-MS/MS confirmed the presence of both hydrophilic and hydrophobic peptides in the complexes with sizes ranging from 275 Da to 1916 Da. Furthermore, reduction in the antioxidant property of peptides following iron complexing indicates iron chelation. Our results suggest that whey protein derived peptide-iron complexes can be used as a potential alternative for chemical iron fortificants for food products and also as iron supplements.

Preparation, characterization and in vitro stability of iron-chelating peptides from mung beans

Mung bean protein was enzymatically hydrolyzed with either alcalase, neutral protease, or papain. The mung bean protein hydrolysates (MPH) showed good ability to chelate ferrous ions, and the chelates had high stability in vitro. The hydrolysates prepared by alcalase showed the highest degree of hydrolysis and the highest ferrous chelating rate. Single factor tests showed that the pH and the material ratio had significant effects on ferrous chelating rates. The optimal MPH to FeCl2·4H2O material ratio was 8:1 (w/w) and the optimal pH of the reaction was 7.0, which yielded a chelating rate of 96.19 ± 0.94%. The fraction 3 with the highest ferrous chelating activity up to 61.25 ± 1.02 μg/mg was obtained from MPH by affinity chromatography. Meanwhile, the MPH-Fe complex had higher digestive stability than just MPH in both in vitro and acid-alkali tolerance assays. The characterization results showed that ferrous ions mainly combined with the amino, carboxyl, imidazole and other chelating active groups in mung bean peptides to form peptide-iron chelates. Scanning electron microscopy (SEM) analysis showed that mung bean peptide chelated ferrous ions to form polymer particles. These results provided insight into ways to develop functional foods such as iron-fortified cereals.

Metabolomics approach based on LC-HRMS for the fast screening of iron(II)-chelating peptides in protein hydrolysates.

Production of iron-chelating peptides from protein hydrolysates requires robust and adequate screening methods to optimize their purification and subsequently valorize their potential antioxidant properties. An original methodology was developed for direct and sensitive screening of iron(II)-chelating peptides based on ion-pair reverse phase liquid chromatography (IP-RPLC) coupled to high-resolution mass spectrometry (HRMS). Peptide mixture was first added to iron(II) solution to form iron(II)-peptide complexes. Then IP-RPLC-HRMS analysis was conducted on this iron-peptide mixture and on the iron-free peptide solution for comparative mass spectra analysis. This protocol, initially applied to a range of low molecular weight standard peptides, allowed detection of [(Peptide-H)+56FeII]+ complex ion for iron(II)-chelating peptides (GGH, EAH, DAH, βAH, DMH, DTH, DSH). GGH was added in complex peptide mixtures and targeted analysis of [(GGH-H)+56FeII]+ complex showed a limit of detection (LOD) below 0.77mgL-1 of GGH. This protocol was finally tested in combination with metabolomics software and additional digital processing for non-targeted search for iron(II)-chelating peptides. Applicability of this new screening methodology has been validated by detection of GGH as iron(II)-chelating peptide when added at 0.77mgL-1 in casein hydrolysate. Graphical abstract.

Evaluation of iron-binding capacity, amino acid composition, functional properties of Acetes japonicus proteolysate and identification of iron-binding peptides

Recovery of functional iron-binding protein hydrolysate from Acetes japonicus employing enzymatic hydrolysis and iron-chelating peptide identification were conducted in this study. The result showed that under the optimal hydrolysis condition including Flavourzyme, pH 5, 50 °C, E:S ratio of 27.4 U/g protein and hydrolysis time of 4.8 h, the obtained proteolysate displayed the maximal iron-binding capacity (IBC) of 177.7 μgFe2+/g protein and comprehended 38,77 % of essential amino acids. Functional features of the Acetes proteolysate encompassing solubility, heat stability, foaming and emulsifying property, oil and water holding capacity were also noteworthy. Peptide fractionation was performed using ultrafiltration and the 1−3 kDa fraction expressed the highest IBC of 120.43 ± 0.15 μgFe2+/g protein, 13.7 times lower than that of disodium ethylenediaminetetraacetate (Na2EDTA). From this fraction, two iron-binding peptides of DSVNFPVHGL (1083.53 Da) and FKVGQENTPILK (1372.77 Da) were identified utilizing nano-UHPLC-MS/MS as well as their de novo spatial structures and interaction with ferrous ion were simulated by PEP-FOLD 3. As a whole, the proteolysate/peptides could be filled as an iron chelator which could shield human body from iron deficient-related disorders or as a functional proteolysate preparation to upgrade food properties.

Food protein-derived iron-chelating peptides: The binding mode and promotive effects of iron bioavailability.

Iron is known as an essential nutrient in the human body. Insufficient iron uptake is easy to result in iron deficiency anemia, which is a public health problem in both developing and developed countries. Iron, complexed by iron-chelating peptides, have been proposed as a superior candidate to ionized iron for improving iron absorption and bioavailability. Nowadays, as more and more iron-chelating peptides are identified from different food sources, the iron-peptide binding mode attracts much attention. Meanwhile, many studies have been conducted on the iron-chelating peptides to make clear their iron bioavailability enhancement potential. The aim of this review is to provide an overview of research progress in food protein-derived iron-chelating peptides. The review would be of particular value for iron-peptide complexes as functional ingredients or iron supplements.

A peptide-Fe(II) complex from Grifola frondosa protein hydrolysates and its immunomodulatory activity

Grifola frondosa protein was hydrolysed using Alcalase, and the resulting hydrolysates (CGFP) were filtered using 5 and 1 kDa nominal cut-off ultrafiltration membranes and separated using Sephadex gel chromatography. Two main fractions, named GFP-1 and GFP-2 were obtained. CGFP-Fe and GFP-Fe were synthesized using ferrous chelation. Results showed that the GFP-2 fraction with molecular weight of 963 Da had the highest Fe(II) chelating activity (2680 ± 40 μg/g). UV spectrum showed that the Fe(II) shifted the maximum absorption to shorter wavelengths. The peptides measured using FTIR showed that the GFP-Fe peptide and the un-chelated peptide had different C–O and C= O absorption peaks. The effects of CGFP, CGFP-Fe, GFP2-2 and GFP-Fe on splenocytes proliferation and cytokines secretion were studied in vitro . Both the GFP-2 and GFP-Fe had good immune-enhancing activity. Furthermore, GFP-Fe showed an ability to promote the proliferation of splenocytes and peritoneal macrophages. The iron-chelating peptide of Grifola frondosa still maintained its immune enhancing activity after in vitro gastrointestinal digestion. The results suggested that GFP-Fe might be beneficial as a new iron supplement and as an immunological enhancement.

Isolation and identification of iron-chelating peptides from casein hydrolysates.

Iron deficiency is a common nutritional disorder worldwide. Peptides derived from protein hydrolysates have recently attracted interest as novel iron chelators due to their superiority in terms of increasing solubility, bioavailability, absorption and stability. The aim of this study was to isolate and identify iron-chelating peptides from casein hydrolysates. Casein was hydrolyzed (trypsin, 3 h) and subsequently isolated using ultrafiltration and RP-HPLC. Four iron-chelating casein hydrolysate peptides, named CHP-1, CHP-2, CHP-3 and CHP-4, were identified by LC-MS/MS, and their amino acid sequences were Glu-Asp-Val-Pro-Ser-Glu-Arg (EDVPSER), His-Lys-Glu-Met-Pro-Phe-Pro-Lys (HKEMPFPK), Asn-Met-Ala-Ile-Asn-Pro-Ser-Lys (NMAINPSK) and Ala-Val-Pro-Tyr-Pro-Gln-Arg (AVPYPQR), with molecular weights of 830.6120 Da, 1012.5280 Da, 873.4440 Da and 829.4570 Da, respectively. The artificially synthesized peptides of CHP-1, CHP-2, CHP-3 and CHP-4 were verified, and their iron-chelating rates were 11.14%, 8.02%, 7.57% and 59.76%, respectively. These results suggested that the isolated iron-chelating peptides might serve as potential iron supplements and be used as food additives and functional foods.

Effect of duck egg white peptide-ferrous chelate on iron bioavailability in vivo and structure characterization.

In order to utilize the industrial by-product 'salted duck egg white' as novel iron additives, the effects of desalted duck egg white peptides-ferrous chelate (DPs-Fe) on the promotion of iron uptake and the structure were investigated. Different doses of DPs-Fe were given and iron sulfate (FeSO4 ) was used as a positive control. After three weeks, hemoglobin (Hb), hematocrit (HCT), red blood cells (RBCs), mean corpuscular volume (MCV), serum iron (SI) and serum ferritin (SF) in iron-deficiency anemia (IDA) rats could be significantly (P < 0.05) increased to the normal levels by DPs-Fe. The gene expressions of divalent metal transporter 1 (DMT1), ferroportin 1 (FPN1) and Hepcidin could be regulated by DPs-Fe. Additionally, DPs-Fe was formed during the chelation process and the structure was characterized. Eight crucial iron-chelating peptides of duck egg white peptides (DPs) were identified by HPLC-ESI-MS/MS, such as Pro-Val-Glu-Glu and Arg-Ser-Ser. It indicated that Glu, Asp, Lys, His, Ser, Cys residues might play crucial roles in the chelating of DPs with iron. DPs-Fe could be a potential iron supplement, and the Glu, Asp, Lys, His played important roles in binding iron and promoting iron uptake. This research expands the understanding of iron uptake by DPs and provides an opportunity for recycling a discarded processing byproduct. © 2018 Society of Chemical Industry.

Identification of iron-chelating peptides from Pacific cod skin gelatin and the possible binding mode

Pacific cod skin gelatin was hydrolyzed under optimized conditions (trypsin, 240min) to generate iron-chelating peptides. Gelatin tryptic hydrolysates were purified by immobilized metal affinity chromatography and reversed phase high performance liquid chromatography. Three novel iron-chelating peptides identified by LC-HRMS/MS were GPAGPHGPPGKDGR, AGPHGPPGKDGR and AGPAGPAGAR, which exhibited high affinity to ferrous ions. The iron-peptide complexation, investigated by ESI-MS and FTIR spectroscopy, showed that the three peptides bound with iron mainly at the ratio of 1:1. Among the groups of the three peptides, the amino and carboxylate terminal groups and peptide bond from peptide backbone, as well as the amino and imine from arginine side chain were involved in the complexation. Moreover, several amino acid side chain groups of GPAGPHGPPGKDGR and AGPHGPPGKDGR, including amino (Lys), imine (His) and carboxylate (Asp), supplied additional iron binding sites. This study suggests a potential application of gelatin-derived peptides as novel carriers to combat iron deficiency.

Separation and identification of iron-chelating peptides from defatted walnut flake by nanoLC-ESI–MS/MS and de novo sequencing

Walnuts are rich in unsaturated fatty acids and are processed to walnut oil. The by-product (defatted walnut flake) consists of approximately 50% protein and is usually treated as waste or low-value-use material. In this study, walnut protein was isolated and hydrolyzed to low-molecular-weight peptides. Iron-binding walnut peptides were prepared by iron-immobilized affinity chromatography (IMAC-Fe3+). Some iron-chelated peptides adsorbed to the IMAC-Fe3+ column were easily broken by NaH2PO4, whereas part of the peptides isolated from IMAC-Fe3+ column remained as peptide-iron complexes and could only be disrupted by EDTA-2Na. Subsequently, the iron-binding walnut peptides were separated into approximately 10 fractions by RP-HPLC. The main peak, with retention time of 32 min, was further analyzed by nanoLC-ESI–MS/MS. Two main components in F32 min were characterized as LAGNPDDEFRPQ and VEDELVAVV with de novo sequencing.

Purification of an iron-chelating peptide from spirulina protein hydrolysates

Iron-chelating peptide was purified from spirulina protein hydrolysates. Spirulina protein was hydrolyzed using Alcalase and Flavourzyme, and the degree of hydrolysis was determined using a trinitrobenzene sulfonic acid assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The spirulina protein hydrolysates were ultra-filtered to isolate the components below 3 kDa, which were then fractionated by Q-Sepharose fast flow and Sephadex G-15 columns. The iron-chelating activity of each fraction was determined, and the peptide with the highest activity was isolated and identified by matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometry. Amino acid sequence of the iron-chelating peptide was identified to be Thr-Asp-Pro-Ile(Leu)-Ala-Ala-Cys-Ile(Leu), which has a molecular weight of 802 Da. Moreover, due to its ability to chelate iron, the isolated peptide could be used as an iron supplement.

Iron-binding properties of sugar cane yeast peptides

The extract of sugar-cane yeast (Saccharomyces cerevisiae) was enzymatically hydrolysed by Alcalase, Protex or Viscozyme. Hydrolysates were fractionated using a membrane ultrafiltration system and peptides smaller than 5kDa were evaluated for iron chelating ability through measurements of iron solubility, binding capacity and dialyzability. Iron-chelating peptides were isolated using immobilized metal affinity chromatography (IMAC). They showed higher content of His, Lys, and Arg than the original hydrolysates. In spite of poor iron solubility, hydrolysates of Viscozyme provided higher iron dialyzability than those of other enzymes. This means that more chelates of iron or complexes were formed and these kept the iron stable during simulated gastro-intestinal digestion in vitro, improving its dialyzability.

Preparation, isolation and identification of iron-chelating peptides derived from Alaska pollock skin

Alaska pollock skin as aquatic by-product was usually discarded in food processing. Iron deficiency has been a nutritional problem for a long period. In this study, Alaska pollock skin was used to generate iron-chelating peptides after being treated by commercial enzymes. Sequential chromatography, including immobilized metal affinity chromatography (IMAC) and reversed phase high performance liquid chromatography (RP-HPLC), was used for capturing and purifying iron-chelating peptides. One tripeptide, with high iron-chelating activity, was obtained and identified by electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS). The amino acid sequence of the iron-chelating peptide was identified to be Ser-Cys-His (MW: 345Da). These results show that Alaska pollock skin could be utilized to generate iron-chelating peptides as an iron supplement in functional food industry.