# Bioaccessibility of iron, zinc, and copper from edible insects available on the European market

M.A.J. Lopes da Costa, Ange Angaïts, Katarzyna Bierla, J.S. de Gois, J. Szpunar

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## To cite this version:

M.A.J. Lopes da Costa, Ange Angaïts, Katarzyna Bierla, J.S. de Gois, J. Szpunar. Bioaccessibility of iron, zinc, and copper from edible insects available on the European market. Applied Food Research, 2025, 5 (1), pp.100918. 10.1016/j.afres.2025.100918. hal-05064477

HAL Id: hal-05064477

https://univ-pau.hal.science/hal-05064477v1

Submitted on 12 May 2025

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# Bioaccessibility of iron, zinc, and copper from edible insects available on the European market

M.A.J. Lopes da Costaª, A. Angaïtsᵇ, K. Bierłaᵇ, J.S. de Goisª ID, J. Szpunarᵇ,ᶜ ID

## ARTICLE INFO

### Keywords:

- Sustainable food sources
- Essential elements
- Gastrointestinal digestion
- SEC
- ICP-MS

## ABSTRACT

Consumption of insects contributes increasingly to sustainable nutrition in the context of global food security. This study investigates the total content and bioaccessibility of essential trace elements Fe, Zn, and Cu in various edible insects. Samples of, among others, mealworms (Tenebrio molitor), silkworms (Bombyx mori), sago worms (Rhynchophorus ferrugineus), crickets (Gryllus bimaculatus), black scorpions (Heterometrus longimanus), and weaver ants (Oecophylla smaragdina) were analysed. Inductively coupled plasma mass spectrometry (ICP-MS) determined significant variability in metal content among different insect species, with an overall content higher than in meat. Bioaccessibility studies were conducted using pepsin, pancreatin and bile salts, to simulate gastrointestinal digestion conditions, as is standard in such studies. The bioaccessibility of Fe, Zn, and Cu ranged from 45 % to 65 % and was generally higher in mealworms than in *Orthoptera* species. Size exclusion chromatography (SEC-ICP-MS) provided insight into the molecular forms of Fe, Zn, and Cu during gastric and intestinal digestion. Intense peaks in the elution range of low molecular mass compounds were observed in the intestinal digests, suggesting complexation of the metal ions with organic ligands. The study highlights the nutritional potential of edible insects as a rich source of essential elements, such as Fe, Zn, and Cu.

## 1. Introduction

Iron, zinc, and copper are essential trace elements crucial for various metabolic processes in the human body. Iron is a component of hae-moglobin, which is responsible for oxygen transport. Its deficiency, commonly associated with anaemia, is a global problem, particularly prevalent in developing countries (Affonfere et al., 2023; European Food Safety Authority, 2015b). Zinc acts as a cofactor for numerous enzymes and plays a vital role in gene expression and cellular regulation. Zinc deficiency poses risks across all age groups, especially for infants and young children, manifesting as impaired growth and other health complications (Affonfere et al., 2023; European Food Safety Authority, 2014; Nosratpour & Jafari, 2018). Copper supports bone formation and myelin sheath development and aids in iron incorporation and absorp-tion. Its deficiency can lead to anaemia and growth impairment, particularly in children (Affonfere et al., 2023; European Food Safety Authority, 2015a).

Protein is essential for the absorption and utilization of iron, zinc, and copper from food. Dietary proteins play a crucial role in binding and transporting these elements through the digestive system. In meat,

proteins facilitate the absorption of the highly bioavailable heme iron (Cabrera & Saadoun, 2014). Similarly, proteins in plant-based foods, such as legumes and grains, aid in the absorption of non-heme iron. Moreover, proteins contain amino acids that act as ligands to chelate and transport zinc and copper in the body, enhancing their bioaccessibility and utilization (Cabrera & Saadoun, 2014). Elements can also be absorbed in ionic form through the paracellular route. Therefore, protein-rich foods in the diet are essential for maintaining the optimal nutritional status of essential nutrients.

The bioaccessibility of trace elements should be considered during the development of novel cost-effective and environmentally friendly food sources. Edible insects, such as crickets, mealworms, and locusts, are rich in protein, iron, zinc, and other essential nutrients, making them a viable alternative to conventional protein sources such as meat and plant-based foods (Halloran et al., 2014; Raheem et al., 2019; van Huis et al., 2013). Insects are highly efficient converters of feed into edible mass, requiring fewer resources to grow compared to traditional live-stock. They produce less greenhouse gas emissions and have a smaller ecological footprint, making them a sustainable option for food pro-duction (Oonincx et al., 2010). Insects can thrive on organic waste

<footnote>ªGraduate Program in Chemical Engineering, Rio de Janeiro State University, Rua São Francisco Xavier 524 – Maracanã, Rio de Janeiro, RJ 20550-013, Brazil</footnote>

<footnote>ᵇCNRS, Institute of Analytical and Physical Chemistry for the Environment and Materials (IPREM), Université de Pau, Hélioparc 2 Av. Pr. Angot, Pau 64053, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: joanna.szpunar@univ-pau.fr (J. Szpunar).</footnote>
materials, contributing to waste reduction and promoting a circular economy (Halloran et al., 2014; Nowakowski et al., 2022). Insect farming requires minimal space and can be undertaken at relatively low cost, making it accessible to small-scale farmers and entrepreneurs (Raheem et al., 2019; Valoppi et al., 2021). Insect-based food products, such as cricket flour or mealworm snacks, contribute to innovation and market diversification in food industry. Insects are culturally accepted as food in many regions of the world, providing a familiar and palatable alternative protein source for consumers (Rumpold & Schlüter, 2013; Valoppi et al., 2021). Although, studies comparing insect species from various geographical regions demonstrate notable disparities in essential element content and emphasize the need for comprehensive characterization and standardization in insect-based food products (Lu et al., 2024). Moreover, the trace element composition varies as a function of insect species, growth phase, and environmental conditions. Larvae generally exhibit higher essential element content than adults (Longvah et al., 2011; Ray & Gangopadhyay, 2021). Element content was shown to depend on the insect colour and habitat (Longvah et al., 2011; Lu et al., 2024). For example, wild *Ruspolia differens* of different colours exhibited differences in element content, underscoring the importance of understanding these variations for nutritional assessment and food production (Lu et al., 2024). Moreover, differences in element composition between reared and wild edible insects of the same species suggest the influence of captivity and environmental factors on nutrient profiles (Lu et al., 2024). Also, study shows that varying the diet of *L. migratoria* leads to significant changes in their element composition, with notable fluctuations in the concentrations of essential elements depending on the feed provided (Oonincx & Van Der Poel, 2011).

While several works reported the trace element content in edible insects, studies of their bioaccessibility have been scarce (Lu et al., 2024; Nowak et al., 2016). Bioaccessibility is defined as the fraction element that is released from the food matrix into the gastrointestinal tract and has the potential to be absorbed. Bioavailability, defined as the proportion of ingested element that are absorbed and available for metabolic functions, varies widely depending on the chemical form of elements in foods, interactions with other components, and physiological factors of consumers (Nosratpour & Jafari, 2018). Consequently, the total element content in food may not directly correlate with the amount of elements absorbed and utilized by the body (Fernández-García et al., 2009; Maares & Haase, 2020).

Bioaccessibility of trace elements is typically evaluated using *in vitro* digestion models, mimicking the physiological conditions of the gastrointestinal tract. Different models have been developed, incorporating sequential digestion steps to assess the breakdown of food matrices and the release of elements (Affonfere et al., 2023; Ojha et al., 2021). Enzymes included α-amylase for oral digestion, pepsin for gastric digestion, and pancreatin for intestinal digestion to simulate nutrient release (simulating the gastrointestinal conditions, not absorption) (Ojha et al., 2021). The static *in vitro* digestion method, adhering to the European BARGE standard, is simple, reproducible, and robust, suitable for mechanistic studies and screenings (Affonfere et al., 2023; Brodkorb et al., 2019). Unlike Caco-2 cell models and dialysis, which simulate absorption rather than gastrointestinal digestion (Affonfere et al., 2023; Maares & Haase, 2020), this method accurately predicts *in vivo* digestion, correlating well with human jejunal digests (Brodkorb et al., 2019). Despite its inability to mimic complex dynamics, it is a reliable tool, extensively used to assess element bioaccessibility in various foods (da Silva et al., 2017; Fernández-García et al., 2009; Gabaza et al., 2018; Hemalatha et al., 2007; Khouzam et al., 2011; Menezes et al., 2018).

The bioaccessibility of elements can be further studied on the molecular level using the coupling of chromatography separating the different chemical forms of metals with element specific detection. Size-exclusion chromatography (SEC) with inductively coupled plasma mass spectrometry (ICP-MS) was the most widely used technique to monitor associations of metal ions with peptides and polysaccharides during simulated gastrointestinal digestion (Wojcieszek et al., 2017; Wojcieszek

& Ruzik, 2016; Dell'Aquila et al., 2020; Gomez et al., 2016; Wang et al., 2019; Kaewkhomdee et al., 2010; Wojcieszek & Ruzik, 2020). Studies of Cu and Zn in various foods of plant origin showed that digestion results in the release of ions and formation of low molecular weight (LMW) complexes, enhancing bioaccessibility (Parzych et al., 2020; Wojcieszek et al., 2017; Wojcieszek & Ruzik, 2016). It was found that Zn in infant formulas was mainly bound to biomolecules of around 10 kDa, indicating high bioavailability, especially in cow milk-based formulas (Gomez et al., 2016).

The growing importance of insect-based foods as source of essential nutrients requires validation, benchmarking and interlaboratory comparability of analytical methods for the determination of the concentration of total elements and their bioaccessibility. National Research Council of Canada (NRCC) recently released certified reference materials of mealworms (VORM-1), crickets (KRIK-1) and black soldier fly (BFLY-1) with the values for total concentrations of several essential trace elements. The goal of this work was to provide data on the bioaccessibility of these elements and an insight into their behaviour in simulated gastrointestinal conditions to be used for benchmarking of the characterization of insect-based foods as source of micronutrients. This task required state-of-the art analytical approaches that have been proposed. An original survey focused on edible insects available on the European market was carried out. The studied species are not only insects but belong to a wider group of arthropods, however, we decided keep the word "insects" which easier identifies the topic of the paper.

## 2. Experimental

### 2.1. Samples

Three mealworm samples at different life stages were obtained directly from the producer, and eleven other samples were purchased from online stores. All samples were already dead, dried, and ready for consumption. They were kept in their original packaging until processed. The samples were ground into a fine, homogeneous powder using a ball mill. After powdering, the samples were stored in appropriate containers under refrigeration (at -20 °C) to preserve their integrity. The Certified Reference Materials (CRMs) used in this study—VORM-1, KRIK-1, and BFLY-1—were also kept refrigerated. All samples were natural insects, without fortification or any added flavors. Information on the samples is presented in Table 1. The CRMs, provided by the National Research Council of Canada (NRCC), were used for validating the total metal determinations.

### 2.2. Instrumentation

A ball mill (MM 200, Retsch, Germany) was used for grinding the insects. pH of buffer solutions was adjusted with a pH meter model FiveEasy (Mettler Toledo, Columbus, OH). A water bath (SBS40, Stuart, UK) was used for incubations during gastrointestinal digestion, water extraction and proteolysis. Centrifugation of the samples was performed using a model 5804 R centrifuge (Eppendorf, Germany). A hot plate (Dig PREP MS, SCP Science, Canada) was used for acid digestion. Total analyses were carried out with an ICPMulti-Quad MS/MS (NexION 5000, Perkin Elmer, Shelton, CT). Chromatograms were obtained using an HPLC 1260 Infinity (Agilent, Santa Clara, CA) coupled with an ICPMS 7700 Series (Tokyo, Japan). The column used was a Superdex™ 75 Increase 10/300 GL (GE Healthcare, Sweden).

### 2.3. Reagents and solutions

All reagents used were of analytical grade unless stated otherwise. Ultrapure water (18 MΩ cm), obtained from a Direct-Q 3UV System (Millipore, Danvers, MA), was used for the preparation of all solutions. For acid digestion nitric acid 67–69 % TraceMetal Grade (Fisher Chemical, Canada) was used. Hydrogen peroxide (30 %) (Baker, The
<caption>**Table 1**</caption>

Samples' description. Code used for each sample, common name of the insect, scientific name of the species, supplier/country of the sample, and stage of life.

<table><thead><tr><th>Code</th><th>Generic name</th><th>Scientific name</th><th>Supplier</th><th>Stage of life</th></tr></thead><tbody><tr><td>VORM-1</td><td>Mealworms</td><td>Tenebrio molitor</td><td>NRC, Canada</td><td>larva</td></tr><tr><td>KRIK-1</td><td>Crickets</td><td>Not informed</td><td>NRC, Canada</td><td>not informed larva</td></tr><tr><td>BFLY-1</td><td>Black soldier fly</td><td>Hermetia illucens</td><td>NRC, Canada</td><td>larva</td></tr><tr><td>A1</td><td>Mealworms</td><td>Tenebrio molitor</td><td>direct producer (Poland)</td><td>12-14 weeks</td></tr><tr><td>A2</td><td></td><td></td><td></td><td>10 weeks adult</td></tr><tr><td>A3</td><td>Black Scorpions</td><td>Heterometrus Iongimanus</td><td>Produced in Thailand by Insectéo,</td><td>larva</td></tr><tr><td>A4</td><td>Weaver ants</td><td>Oecophylla smaragdina</td><td>commercialized in France</td><td>larva</td></tr><tr><td>A5</td><td></td><td></td><td></td><td>larva</td></tr><tr><td>A6</td><td>Silkworms</td><td>Bombyx Mori</td><td></td><td>larva</td></tr><tr><td>A7</td><td>Sago Worms</td><td>Rhynchophorus Ferrugineus</td><td></td><td>larva</td></tr><tr><td>A8</td><td>Morio worms</td><td>Zophobas Morio</td><td></td><td>larva</td></tr><tr><td>A9</td><td>Grasshoppers</td><td>Locusta Migratoria</td><td></td><td>adult</td></tr><tr><td>A10</td><td>Mealworms</td><td>Tenebrio molitor</td><td></td><td>larva</td></tr><tr><td>A11</td><td>Crickets</td><td>Gryllus Bimaculatus</td><td></td><td>adult</td></tr><tr><td>A12</td><td>Water Scorpions</td><td>Nepidae</td><td></td><td>adult</td></tr><tr><td>A13</td><td>Mealworms</td><td>Tenebrio molitor</td><td>Produced in France by Jimini's,</td><td>larva</td></tr><tr><td>A14</td><td>Crickets</td><td>Acheta domesticus</td><td>commercialized in France</td><td>adult</td></tr></tbody></table>

Netherlands) was used during acid digestion. Protease in Trisma buffer (Merck KGaA, Darmstadt, Germany) was used for proteolysis. α-amylase was purchased from Merck-Sigma-Aldrich (Saint-Quentin-Fallavier, France). Buffer for gastrointestinal digestion was prepared using sodium phosphate dibasic (TraceSELECT®, Fluka Analytical, Germany) and pH was adjusted with phosphoric acid 85% (Aldrich Chemistry, Germany). Pepsin from porcine gastric mucosa (P7000, Sigma, MO), pancreatin from porcine pancreas (P7545, Sigma, MO) and bile salt (B8756, Fluka Analytical, New Zealand) were used for gastrointestinal digestion. Chromatographic mobile phase 0.1 mol/L was prepared from ammonium acetate, puriss. p.a. (Sigma-Aldrich, Germany). For calibration curves, Fe, Zn, and Cu standard 1000 mg/L solutions (Analytichem, Quebec, Canada) were diluted as necessary.

## 2.4. Procedures

### 2.4.1. Acid digestion

Approximately 0.2 g of powdered sample was weighed, and 2 mL of conc. nitric acid was added. The mixture was then placed on a hot plate at 65 °C for 4 h. Subsequently, 0.5 mL of H₂O₂ was added to the solution, followed by another 4 h on the hot plate at the same temperature. The acid digestion of the extracts was carried out using 0.25 mL of the extract, 1.0 mL of conc. HNO₃, and 0.5 mL of H₂O₂. The solution was placed on a hot plate at 65 °C for 5 h. After digestion, the sample volume was made up to 10 mL.

### 2.4.2. Water extraction, proteolysis and gastrointestinal in vitro digestion

0.2 g of the sample was weighed, suspended in 5 mL of water placed in an ultrasonic bath at room temperature for 1 h. The sample was centrifuged. The supernatant was separated for further analysis by centrifugation, while the residue was used for proteolysis. For proteolysis, 5 mL of Tris buffer (30 mmol/L) and 20 mg of protease were added to the residue after water extraction. The mixture was incubated in a water bath at 37 °C for at least 17 h. Then, the mixture was centrifuged, and the supernatant was collected for further analysis.

In vitro gastrointestinal digestion was conducted according to Ma et al. (2020), with some modifications. In brief, 0.1 g of sample was weighed into a centrifuge tube. During the gastric digestion step, it was placed in 8.5 mL HCl (pH 2,) and 1 mL of gastric digestion buffer (18,000 U/mL pepsin in 0.1 mol/L HCl, pH 2.0) was added and the mixture was incubated at 37 °C for 1 h with mechanical agitation. Subsequently, the mixture was centrifuged, and the supernatant was separated for further analysis. The residue after the gastric digestion step was mixed with phosphate buffered saline (PBS) (pH 7.4, 8.5 mL) and small intestinal digestion buffer (2 mg pancreatin and 25 mg bile in 1 mL PBS, pH 7.4, 1 mL) and incubated at 37 °C for 2 h with mechanical agitation. The mixture was centrifuged again to separate the supernatant for further analysis. The concentrations of the elements in the supernatants from both the gastric and intestinal phases were measured. The bio-accessibility was calculated as the percentage of the total element content in the combined supernatants relative to the content in the original samples (w/w, dry mass).

### 2.4.3. Instrumental analysis

For the total metal analysis, the sample digest was diluted, depending on the expected concentration of the analytes, typically 250–1000 times. The samples were analysed by ICP-MS using a collision cell pressurised with O₂ (0.8 mL/min), H₂ (1.15 mL/min), and NH₃ (1 mL/min) gas. The operating parameters were set as follows: radio frequency power of 1600 W, auxiliary gas flow rate of 1.2 L/min, and nebulizer gas flow rate of 1.0 L/min. The isotopes monitored included ⁵⁷Fe, ⁵⁶Fe, ⁵⁴Fe, ⁶⁶Zn, ⁶⁴Zn, ⁶⁵Cu, and ⁶³Cu. Digests were analysed in triplicate.

To ensure quality control, CRMs were used. Analytical blanks were run in parallel. Quantification was conducted by external calibration, within the range of 0.05–15 ppb. Values for the total analysis are reported as mean ± SD of three true analytical replicates. Values for the extracts are reported as mean ± SD for two replicates.

For SEC separations, the supernatant after extraction was injected directly or upon a dilution, with water, ranging from 10 to 25 times, when necessary. Elution was isocratic at 0.8 mL/min with 100 mM ammonium acetate (pH 7.5). Injection volume of 100 µL. The exit of the column was directly connected to an ICP-MS equipped with a reaction cell. Helium and H₂ gas were used at 1 mL/min and 3.5 mL/min gas flow rate, respectively. Nickel cones were used. The operating parameters were set as follows: radio frequency power of 1550 W, makeup gas flow rate of 0.1 L/min, and carrier gas flow rate of 1.0 L/min. The isotopes monitored were ⁵⁶Fe, ⁵⁴Fe, ⁶⁶Zn, ⁶⁴Zn, ⁶⁵Cu, and ⁶³Cu.

# 3. Results and discussion

## 3.1. Characterization of the binding of metals in CRMs

In order to provide reference data for the methodology to be used for the investigation of insect-based foods available on the European market, certified reference materials, with a similar matrix were studied. Firstly, the total concentrations of iron, zinc, and copper were determined and they agreed with the certified values within the uncertainty range given by NRCC. The results can be found in Table SI-1 with their respectively zeta score (according to the ISO 13528 guideline). The parameters investigated subsequently included the extraction recovery of trace elements as well as fractionation according to molecular mass by size-exclusion chromatography.

### 3.1.1. Effect of the extraction procedure

The water-soluble fraction constitutes the potentially most easily available part of the metal content but also that can be prone to losses during food processing. On the other hand, proteolysis involves breaking down proteins into amino acids and peptides, and the concentration of metal extracted by proteolysis indicates the amount of metal bound to proteins within the food matrix. When carried out on the water extraction residue, it refers to the insoluble protein fraction. A
preliminary study was conducted to assess both the water-soluble fraction and water-insoluble protein-bound fraction of Fe, Zn, and Cu in the CRMs. The results are presented in Fig. 1.

The results reveal that the water-soluble fraction of the samples is low, particularly for Fe and Zn. However, it is important to note that the bioaccessibility of these metals depends not only on their solubility but also on their chemical form. For instance, heme iron, which is found in animal tissues, has a higher bioavailability than non-heme iron and may help explain the results obtained, particularly the low water-soluble fraction of iron. Additionally, copper exhibited the highest water-soluble and protein-bound fraction among all samples. Iron and Zn results were quite similar, although Zn content was slightly higher in the black soldier fly sample, while iron was slightly higher in the cricket flour sample. However, the number of samples studied does not allow for a comprehensive statistical confirmation of these observations. Furthermore, it should be considered that the release of metals is also influenced by factors such as the ionic strength of the solution, which was not simulated in the digestion process used in this study. The fraction of metal that remains unextracted by both proteolysis and water suggests reduced bioaccessibility of metal ions in these conditions.

Fig. 2 presents the data on the recovery using solutions mimicking digestion conditions in the mouth (α-amylase), gastric tract (pepsin) and intestine (pancreatin). They indicate minimal metal release during the oral digestion phase, with a notable increase during the gastric phase. Specifically, Cu appears to become more available during the intestinal phase of digestion. The overall recovery reaches or exceed 80 % with the exception of iron in the BFLY-1 material.

### 3.1.2. Size-exclusion chromatography of CRMs

In order to confirm the release of trace elements and get an insight into their chemical forms the gastric and the subsequent intestinal digests were analysed by SEC-ICP-MS. The morphology of the chromatograms for all the three materials analysed was found to be similar and the example chromatograms are presented in Fig. 3 (for the VORM-1 material).

For gastric digests the chromatograms show signals in the LMW range, as the resolved peaks (in the case of Zn at 23 and 27 min) or a cluster of peaks (for Fe and Cu) centred at 23 min and coeluting with the first peak observed for iron. For the subsequent intestinal step, an additional peak is seen in the void of the column for all the three elements.

The morphology of the chromatograms is characteristic of the products of autolysis of proteases acting as ligands for the trace element ions released from the sample. The excess of these ligands can overlap

with those released from the samples, as indicated earlier by Parzych et al. (2020), who observed a significant contribution of the blank. Indeed, enzymes present in cocktails used in the simulated digestion schemes (and the products of their autolysis) have the ability to complex metal ions released in the digestion process, producing chromatographic peaks that are difficult to distinguish from those resulting from the food matrix. We can conclude that the studied metals released during the gastrointestinal digestion process are present in the form of a potentially easily bioavailable mixture of low molecular weight species containing complexes with both digested food constituents and autodigested enzymes. The morphology of the chromatograms is essentially similar to those reported earlier for the bioaccessibility of metals from plants (Parzych et al., 2020; Ruzik & Wojcieszek, 2016; Wojcieszek et al., 2017) and Zn from milk (Gomez et al., 2016).

## 3.2. Characterization of the marketed insect-based food samples

### 3.2.1. Total concentrations of Fe, Zn and Cu

Table 2 summarizes the total concentrations of Fe, Zn, and Cu found. The first group is worms, which include mealworms from different suppliers or developmental stages (A1 to A3, A10, A13), as well as silkworms (A6), sago worms (A7), and morio worms (A8). The concentrations found fall within the range reported in literature (Lu et al., 2024; Nowak et al., 2016; Papastavropoulou et al., 2022). The silkworm sample (A6) showed Fe, Zn, and Cu concentrations consistent with literature findings for Bombyx Mori (Köhler et al., 2019; Longvah et al., 2011; Lu et al., 2024), with notably higher Zn levels compared to other silkworm species (Ray & Gangopadhyay, 2021; Zhou & Han, 2006) and the other worm samples. The sago worm sample (A7) showed a lower concentration of Fe compared to the other worm samples, although the results align with those reported in the literature for the same species (Rhynchophorus Ferrugineus) (Kavle et al., 2023). Also, it contained higher concentrations than other sago worm species (Kavle et al., 2023; Köhler et al., 2020; Lu et al., 2024). The morio worm sample (A8) contained less Zn and Cu but the results were similar to literature data for Zophobas Morio (Lu et al., 2024).

The total Fe, Zn, and Cu concentrations were determined in three *Orthoptera* species: *Locusta Migratoria* (A9), *Gryllus Bimaculatus* (A11), and *Acheta domesticus* (A14). The results fall within the reported literature range (Köhler et al., 2019; Kosecková et al., 2022; Lu et al., 2024; Mwangi et al., 2018; Papastavropoulou et al., 2022). *Locusta migratoria* exhibited elevated concentrations of Cu compared to other *Orthoptera* species. This species was shown to have the capability to chelate Cu²⁺ ions, and it was observed that, in certain samples, the concentration of

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Relative Fe, Zn and Cu content in water soluble (orange) and (water insoluble) protein (blue) fractions of KRIK-1, BFLY-1 and VOM-1 standard reference materials (NRCC, Canada).</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Fig. 2. Relative content of Fe, Zn and Cu released during simulated sequential digestion: oral (marine blue), gastric (light blue) and intestinal (green) of KRIK-1, BFLY-1 and VOM-1 standard reference materials (NRCC, Canada).</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>Fig. 3. Example of the molecular size-distribution of Fe, Zn, and Cu species in simulated gastric (upper panel) and intestinal (bottom panel) digestates of insect CRMs probed by SEC-ICP-MS (shown for BFLY-1).</figcaption></figure>

<caption>**Table 2**</caption>

Total content (µg/g of dry matter) of Fe, Zn, and Cu in the samples organized by group. And results of the CRM recovery conducted concurrently with the samples.

<table><thead><tr><th rowspan="2">Group</th><th rowspan="2">Species</th><th rowspan="2">Code</th><th colspan="2">Fe</th><th colspan="2">Zn</th><th colspan="2">Cu</th></tr><tr><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th></tr></thead><tbody><tr><td rowspan="6">Worms</td><td rowspan="5">Tenebrio Molitor</td><td>A1</td><td>94.4</td><td>7.22</td><td>151.1</td><td>7.81</td><td>14.0</td><td>0.41</td></tr><tr><td>A2</td><td>69.8</td><td>4.94</td><td>151.4</td><td>3.84</td><td>19.6</td><td>0.76</td></tr><tr><td>A3</td><td>52.1</td><td>0.43</td><td>103.5</td><td>4.02</td><td>13.5</td><td>0.81</td></tr><tr><td>A10</td><td>45.9</td><td>3.8</td><td>115.4</td><td>9.57</td><td>18.4</td><td>1.37</td></tr><tr><td>A13</td><td>49.1</td><td>0.54</td><td>112.8</td><td>2.43</td><td>15.0</td><td>0.3</td></tr><tr><td>Bombyx Mori</td><td>A6</td><td>51.1</td><td>4.59</td><td>190.4</td><td>1.74</td><td>11.5</td><td>0.36</td></tr><tr><td rowspan="3">Orthoptera</td><td>Rhynchophorus Ferrugineus</td><td>A7</td><td>28.1</td><td>7.02</td><td>108.0</td><td>2.14</td><td>10.61</td><td>0.75</td></tr><tr><td>Zophobas Morio</td><td>A8</td><td>47.4</td><td>3.33</td><td>72.9</td><td>3.54</td><td>8.52</td><td>0.3</td></tr><tr><td>Locusta Migratoria</td><td>A9</td><td>97.7</td><td>5.46</td><td>182.1</td><td>4.45</td><td>102.1</td><td>1.61</td></tr><tr><td rowspan="2">Others</td><td>Gryllus Bimaculatus</td><td>A11</td><td>60.7</td><td>0.17</td><td>164.0</td><td>14.8</td><td>20.4</td><td>0.49</td></tr><tr><td>Acheta domesticus</td><td>A14</td><td>100.4</td><td>2.48</td><td>240.6</td><td>7.96</td><td>32.0</td><td>0.23</td></tr><tr><td rowspan="2"></td><td>Heterometrus longimanus</td><td>A4</td><td>108.8</td><td>8.82</td><td>630.0</td><td>27.5</td><td>131.0</td><td>1.87</td></tr><tr><td>Oecophylla smaragdina</td><td>A5</td><td>194.1</td><td>2.92</td><td>89.48</td><td>1.26</td><td>21.6</td><td>0.56</td></tr><tr><td colspan="2">CRM Recovery (%)</td><td>A12</td><td>218.6</td><td>5.31</td><td>133.3</td><td>6.43</td><td>14.8</td><td>0.55</td></tr></tbody><tfoot><tr><td colspan="2"></td><td></td><td>101.7 ± 9.9</td><td></td><td>82.8 ± 2.3</td><td></td><td>85.0 ± 2.9</td><td></td></tr></tfoot></table>
Cu approached levels considered as toxic (Acosta-Estrada et al., 2021; Murefu et al., 2019). The diet clearly affects the nutritional composition of the species. When the diet of *L. migratoria* was changed, concentrations of Fe, Zn, and Cu ranged from 78 to 217 mg/kg, 137 to 172 mg/kg, and 24 to 41 mg/kg, respectively (Oonincx & Van Der Poel, 2011). The highest Cu concentration was for a diet of grass and wheat bran and adults, and the lowest for a diet of grass and penultimate instars. For Fe, the highest was with a diet of grass and wheat bran and adults, and the lowest was with a diet of grass, wheat bran and carrots and penultimate instars. And for Zn, the largest was with a diet of grass and adults, and the smallest was a diet of grass and wheat bran and penultimate instars.

*Gryllus Bimaculatus* showed lower concentrations of Fe, Zn, and Cu compared to the *Orthoptera* species analysed in this work. However, the concentrations found in our study are similar to other reports of Fe concentrations (97–129 µg/g), Zn concentrations (224–321 µg/g), and Cu concentrations (32–98 µg/g) (Lu et al., 2024; Mwangi et al., 2018; Ojha et al., 2021).

*Acheta domesticus* is one of the most widely consumed insects in various regions. Studies showed concentrations of Fe, Zn, and Cu in the range of 12–197 µg/g, 116–217 µg/g, and 9–30 µg/g, respectively (Köhler et al., 2019; Kosečková et al., 2022; Lu et al., 2024; Ojha et al., 2021). The concentrations found here are generally similar to the reported values, although there is notable variability, particularly in the case of Fe. A recent review indicates that the diet of this species significantly influences its final nutritional composition (Lu et al., 2024).

The total concentrations of Fe, Zn, and Cu were also determined in samples of black scorpion, weaver ants, and *Nepidae*. The black scorpion exhibited the highest concentrations of Zn and Cu. *Nepidae* showed the highest concentration of Fe, followed by weaver ants. Taxonomically, scorpions are not classified as insects but belong to the same *phylum*, *Arthropoda*. The concentrations found are higher than those reported elsewhere for black scorpion (26, 33, and 57 ppm, respectively, for Fe, Zn, and Cu) (Aydogan, 2021). Due to the scarcity of studies on scorpion elemental composition, the reasons for these differences remain unclear. Regarding weaver ants, a study reported concentrations of Fe, Zn, and Cu at 156.6 µg/g, 189.7 µg/g, and 8.5 µg/g, respectively (Chakravorty et al., 2016). In our study, higher concentrations of Fe and Cu were found, but the concentration of Zn was lower.

Table 3 shows that the concentrations of Fe, Zn, and Cu found in edible marketed insects are higher than those reported recently in various types of traditional cooked meats (USDA, 2024). Consequently, insects emerge as a significant source of Fe, Zn, and Cu, especially when compared to traditional meat food. Even when compared to the liver, considered as metal accumulator, the insects are only lower in Cu content. Consumption of a 10 g portion of insects can provide varying amounts of Fe, Zn, and copper Cu depending on the type of insect. Considering the three groups of insects presented, the contribution of iron ranges from 0.281 mg to 2.186 mg. With a daily recommendation of 8 mg for adult men (EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), 2015), this portion would provide between 3.5 % and

<caption>**Table 3**</caption>

Total content of Fe, Zn, and Cu in different types of cooked meat (µg/g) compared to the mean of content found in this study.

<table><thead><tr><th></th><th>Meat type</th><th>Fe</th><th>Zn</th><th>Cu</th></tr></thead><tbody><tr><td rowspan="5">U.S.D.A. data on traditional meat</td><td>beef (loin)</td><td>30.4</td><td>37</td><td>0.97</td></tr><tr><td>beef (liver)</td><td>65.4</td><td>53</td><td>143</td></tr><tr><td>pork (cured)</td><td>14.9</td><td>33.6</td><td>1.82</td></tr><tr><td>chicken (meat)</td><td>4.9</td><td>9.6</td><td>0.44</td></tr><tr><td>fish (tilapia)</td><td>6.9</td><td>4.1</td><td>0.75</td></tr><tr><td rowspan="4">This study (by group)</td><td>worms</td><td>28.1 - 94.4</td><td>72.9 - 190.4</td><td>8.5 - 19.6</td></tr><tr><td>Orthoptera</td><td>60.8 - 100.4</td><td>164 - 240.6</td><td>20.4 - 32.0</td></tr><tr><td>others</td><td>108.8 - 218.6</td><td>89.5 - 630</td><td>14.8 - 131</td></tr></tbody></table>

27.3 % of the recommended daily intake of iron. For zinc, the intake ranges from 0.895 mg to 6.3 mg, corresponding to 8.1 % to 57.3 % of the recommended daily intake of 11 mg for men (EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), 2014). As for copper, with concentrations ranging from 0.085 mg to 1.31 mg, it would provide between 9.4 % and 145.6 % of the daily recommended value of 0.9 mg (Institute of Medicine (US), 2001). These results indicate that even small portions of insects, such as 10 g, can significantly contribute to the daily intake of essential elements, especially zinc and copper.

### 3.2.2. Bioaccessibility of Fe, Zn and Cu

As the extraction recovery by simulated gastric juice was similar to the final extraction recovery when the gastric digestion step was preceded by simulated oral digestion step, the latter was omitted. Fig. 4 summarizes the percentage of Fe, Zn and Cu released in the gastric and intestinal steps, the sum of which is a measure of bioaccessibility. Food matrix composition and interactions with other components affect the bioaccessibility of nutritional compounds (Ojha et al., 2021). The bioaccessibility ranged from 45 % to 65 % for Fe, Zn, and Cu. Samples from the worm group tended to have higher bioaccessibility compared to *Orthoptera* samples. These values are similar to the values reported for beef and lamb meat for Fe (60–70 %), Zn (40–50 %), and Cu (≤ 40 %) (Cabrera & Saadoun, 2014).

The similar bioaccessibility of Zn in both insect and meat samples is a significant finding given the essential role of this element in human nutrition. Conversely, the bioaccessibility of Fe was slightly lower in the insect samples. Non-haem iron, predominant in most edible insects, may exhibit poor solubility under intestinal conditions and could be influenced by other dietary components (Mwangi et al., 2018).

Although bioaccessibility studies of micronutrients in edible insects are limited, proteins and amino acids such as histidine and methionine have been recognized for enhancing the absorption of non-haem iron and zinc, suggesting that insects with higher protein content may facilitate element uptake (Mwangi et al., 2018). The bioaccessibility of Cu appears to be higher in insects compared to meat.

Note that processing can significantly impact nutrient bioaccessibility in insects, with studies showing that changes in the treatment of *T. Molitor* resulted in varying bioaccessible Zn levels from 20 % to 40 % (Kröncke et al., 2019). Zinc and iron are notably sensitive to processing, and high levels of insoluble dietary fibre can also reduce the bioaccessibility of these elements (Matiza Ruzengwe et al., 2022).

It's important to highlight that although insects and meat seem to exhibit similar bioaccessibility, insects possess higher overall concentrations of essential nutrients, such as iron, copper, and zinc. Consequently, insects emerge as richer sources of these nutrients.

### 3.2.3. SEC-ICP-MS of gastrointestinal digests

The molecular size-distribution was probed by SEC-ICP-MS for one sample from each studied group (worms, *Orthoptera*, and black scorpion) in the gastric and the intestinal digests. The chromatograms for Fe, Zn, and Cu for the studied samples were very similar so Fig. 5 shows only one set of them (for worms) with the remaining ones shown in Fig. SI-2. The Fe and Zn peaks observed during the analysis of intestinal digest appear at the same elution times as one of the peaks in gastric digest; for Cu both digests present a broad peak most probably corresponding to several unresolved species. Fe, Zn, and Cu are associated with molecules smaller than 300 Da. A notable peak at 10 min was observed only in the intestinal phase for Fe, suggesting that Fe may be bound to a molecule larger than 70 kDa, which becomes accessible primarily in the intestinal step. The overall morphology of the chromatograms is very similar to that shown for certified reference materials in Fig. 3 which is partly due to the abundance of ligands resulted from the autolysis of the enzymes. The chromatograms in Fig. 4 prove that the enzymolysis of insect foods leads to the release of metal ions which are efficiently complexed in gastrointestinal conditions and thus bioaccessible.

In insects, the possible forms of Fe, Zn, and Cu can vary based on
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Relative content of Fe, Zn and Cu released during simulated sequential digestion: gastric (marine blue) and intestinal (light blue) of studies samples of edible insects (cf. Table 1).</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Example of the molecular size-distribution of Fe, Zn, and Cu species in simulated gastric (upper panel) and intestinal (bottom panel) digestates of insects probed by SEC-ICP-MS (shown for sample A13, mealworm).</figcaption></figure>

their physiological roles and interactions within the insect's body. In vertebrates, iron is commonly associated with myoglobin and haemo-globin in muscles, but in insects, iron is predominantly found in cyto-chromes (Mwangi et al., 2018). Cytochromes containing haem-Fe are presumed to have similar bioaccessibility to myoglobin and haemoglo-bin. Additionally, iron in insects is largely present as non-haem mole-cules such as ferritin and holoferritin, acting as storage proteins for iron. Each ferritin molecule can hold thousands of iron atoms, primarily in the ferrous state, enhancing their bioaccessibility. Although insect ferritin differs from vertebrate ferritin, its precise characteristics are not yet fully understood (Mwangi et al., 2018). All these molecules are degraded in the presence of proteolytic enzymes. Zinc and copper in insects can be complexed with various molecules but, to the best of our knowledge, no information about their forms in insects is available.

## 4. Conclusion

This study of trace elements in various insect species, evaluated as potential sustainable food sources in the European Union, reveals con-centrations of Fe, Cu, and Zn that surpass those typically found in red meat, indicating insects as a promising source of essential trace elements with high bioaccessibility. The investigation of molecular weight dis-tribution suggests the presence of these elements in the gastrointestinal digest in low molecular weight compounds, likely to be small peptides. By characterizing CRMs specifically designed for edible insect samples, in terms of fractionation of metal species, this research facilitates the standardization and contributes to the improvement of accuracy of

analytical techniques, advancing the field of insect-based food analysis and supporting future investigations into sustainable food sources.

### Declaration of ethical statement - studies in humans and animals

The authors declare that the submitted paper reports no studies in humans and animals.

### CRediT authorship contribution statement

M.A.J. Lopes da Costa: Writing – original draft, Investigation, Formal analysis. A. Angaïts: Validation, Methodology, Formal analysis. K. Bierla: Validation, Methodology. J.S. de Gois: Funding acquisition. J. Szpunar: Writing – review & editing, Validation, Supervision, Resources, Conceptualization.

### Declaration of competing interest

The authors declare the following financial interests/personal re- lationships which may be considered as potential competing interests: Marina A. J. Lopes da Costa reports financial support was provided by Coordination of Higher Education Personnel Improvement. Marina A. J. Lopes da Costa reports a relationship with Carlos Chagas Filho Foun- dation for Research Support of Rio de Janeiro State that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
## Acknowledgments

The first author is thankful to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) [n° 88881.846769/2023-01] for a travel grant and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) - Finance Code 001. Fundação Carlos Chagas de Amparo à Pesquisa do Estado do Rio de Janeiro - FAPERJ, and Universidade do Estado do Rio de Janeiro for their financial support. A gift of the standard reference materials from the Inorganic Chemical Metrology laboratory of the NRC, Canada, is appreciated.

## Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.afres.2025.100918.

## Data availability

Data will be made available on request.

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