The Influence of Ligands on the Pd-Catalyzed Diarylation of Vinyl Esters.

The impact of ligands on the palladium-catalyzed 1,2-diarylation reaction course is presented. The application of Pd-dmpzc as a catalyst provides an efficient, chemoselective and sustainable protocol for the synthesis of valuable 1,2-diphenylethyl acetates. The reaction is conducted in water under mild conditions. Reaction products can be easily separated from the reaction mixture and catalyst by simple extraction. What is more, the rational choice of catalyst significantly reduces the leaching of the metal into the product and its contamination (0.1 ppm). Efficient phase separation and ultralow Pd leaching enable the reuse of the water medium containing the Pd-dmpzc catalyst several times without a significant loss of activity and with even higher selectivity (from 95% to 100% in the third cycle). The recyclability of both the catalyst and the reaction medium together with high chemoselectivity and low palladium leaching reduces the amount of waste and the cost of the process, exhibiting an example of a sustainable and green methodology.

Fe3O4SPIONs in cancer theranostics-structure versus interactions with proteins and methods of their investigation.

As the second leading cause of death worldwide, neoplastic diseases are one of the biggest challenges for public health care. Contemporary medicine seeks potential tools for fighting cancer within nanomedicine, as various nanomaterials can be used for both diagnostics and therapies. Among those of particular interest are superparamagnetic iron oxide nanoparticles (SPIONs), due to their unique magnetic properties,. However, while the number of new SPIONs, suitably modified and functionalized, designed for medical purposes, has been gradually increasing, it has not yet been translated into the number of approved clinical solutions. The presented review covers various issues related to SPIONs of potential theranostic applications. It refers to structural considerations (the nanoparticle core, most often used modifications and functionalizations) and the ways of characterizing newly designed nanoparticles. The discussion about the phenomenon of protein corona formation leads to the conclusion that the scarcity of proper tools to investigate the interactions between SPIONs and human serum proteins is the reason for difficulties in introducing them into clinical applications. The review emphasizes the importance of understanding the mechanism behind the protein corona formation, as it has a crucial impact on the effectiveness of designed SPIONs in the physiological environment.

The microalgae's ability to accumulate selected trace elements studied by ICP-MS/MS and chemometric methods.

BACKGROUND: Microalgae can be used in different branches of industry, including cosmetology, pharmaceutics and the food industry, information on their ability to accumulate different elements becomes more important. The microalgae biomass grown in the media enriched in elements can increase the accumulation of different ions and give a possibility to control the contents of the various elements. METHODS: The aim of the study was to determine the total content of metals in microalgae by tandem mass spectrometry with inductively coupled plasma (ICP-MS/MS) and analysis of the contents of particular metals as a function of the type of microalgae and conditions of cultivation. As the adverse effects of metals on the health of humans and animals have been well-documented and the use of microalgae has increased, the knowledge of metal contents in them is of particular importance in control of their quality. RESULTS: Analysis of results permitted distinction of three main groups of microalgae with similar total metal content levels. Moreover, the results revealed the ways of stimulating more significant accumulation of selected elements (for example, Se concentration in control algae 0.279 µg g-1, in the algae cultivated in enriched medium - 219.7 µg g-1). They indicated the possible correlations between the accumulation of different ions. The result obtained shows a significant effect of metal accumulation and has a considerable impact on the differentiation of Arthrospira platensis grown in the medium enriched in different elements (selenium, zinc, chromium) (p ≤ 0.05). CONCLUSIONS: Particular impact on the content of selected elements had the conditions of cultivation (type of support) and the microalgae species. Although the one species as the most significant source of selected elements cannot be indicated, it is possible to control the accumulation by the composition of the medium.

Nanoscale Ion-Exchange Materials: From Analytical Chemistry to Industrial and Biomedical Applications.

Nano-sized ion exchangers (NIEs) combine the properties of common bulk ion-exchange polymers with the unique advantages of downsizing into nanoparticulate matter. In particular, being by nature milti-charged ions exchangers, NIEs possess high reactivity and stability in suspensions. This brief review provides an introduction to the emerging landscape of various NIE materials and summarizes their actual and potential applications. Special attention is paid to the different methods of NIE fabrication and studying their ion-exchange behavior. Critically discussed are different examples of using NIEs in chemical analysis, e.g., as solid-phase extraction materials, ion chromatography separating phases, modifiers for capillary electrophoresis, etc., and in industry (fuel cells, catalysis, water softening). Also brought into focus is the potential of NIEs for controlled drug and contrast agent delivery.

Enhanced edible plant production using nano-manganese and nano-iron fertilizers: Current status, detection methods and risk assessment.

BACKGROUND: Nanotechnology offers many benefits in the globally important field of food production and human nutrition, particularly by implementing agricultural nanoproducts. Of these, edible plant fertilizers enriched with nanosized forms of essential metals, Mn and Fe, are growing in importance with the advantages of enhanced action on plant roots. SCOPE AND APPROACH: This review focuses on the importance of tracking the bioaccumulation and biodistribution of these pertinent nanofertilizers. An emphasis is given to the critical analysis of the state-of-the-art analytical strategies to examine the Mn and Fe nanoparticles in edible plant systems as well as to shedding light on the vast gap in the methodologies dedicated to the speciation, in vitro simulation, and safety testing of these promising nanomaterials. Also provided are guidances for the food chemists and technologists on the lights and shadows of particular analytical approaches as a matter of authors' expertise as analytical chemists. KEY FINDINGS AND CONCLUSIONS: While the use of nanotechnology in agriculture seems to be growing increasingly, there is still a lack of analytical methodologies capable of investigating novel Mn- and Fe-based nanomaterials as potential fertilizers. Only the advent of reliable analytical tools in the field could bridge the gaps in our knowledge about processes in which those materials participate in the plant systems and their effects on crop production and quality of the produced food.

Drawbacks in the efficient monitoring of gold nanoparticle-based cisplatin delivery systems formation by HPLC-ICP-MS.

Since chemotherapy suffers many limitations related to side effects of anticancer drugs (e.g. cisplatin - CDDP), nanoparticles are probed as carriers in targeted drug delivery. Gold nanoparticles (AuNPs) are broadly investigated due to their biocompatibility, nontoxicity, and tunable surface. Despite many AuNPs-cisplatin systems (AuNP-CS) reports found in the literature, only a few include studies of their synthesis and formation efficiency using analytical tools providing simultaneously qualitative and quantitative analytical information. Therefore, this research continues our previous study of AuNP-CS formation investigated by capillary electrophoresis with inductively coupled plasma mass spectrometry (ICP-MS). Namely, it presents the analogical approach but employs the coupling of another separation technique: isocratic reversed-phase high-performance liquid chromatography. The study concerns the difficulties of analytical method optimization path and contains a discussion of the observed problematic issues related to the analysis and preparation of AuNP-CS. Moreover, the presented work confronts the performance and applicability of both tools for the scrutiny of AuNP-CS, especially considering the comparison of their resolution power.

Speciation of Arsenic(III) and Arsenic(V) in Plant-Based Drinks.

Recently, food products based only on plants have become increasingly popular and are often found on store shelves. It is a specific market response to the growing demand for, and interest in, plant foods. Cow's milk has also gained its counterpart in the form of plant-based beverages, based on cereals, nuts or legumes. The emergence of an increasingly wide range of plant-based food products has also led to increased research on safe plant food consumption. This study was conducted to quantify total arsenic content and its species (arsenic(III) and (V)) in samples of plant-based beverages purchased at Polish markets. Speciation analysis of arsenic was performed by high-performance liquid chromatography combined with inductively coupled plasma mass spectrometry. The presented study was conducted on six selected plant-based beverages, including almond, millet, soybean, rice, coconut and oat. An analysis using size exclusion chromatography was performed. In order to initially visualize the content of the observed elements and the particle size of the compounds in which they occur, at first the samples were subjected to the size-exclusion chromatography. Speciation analysis of arsenic was carried out using anion-exchange liquid chromatography, combined with inductively coupled plasma mass spectrometry. The presented method was validated with certified reference material (CRM rice flour).

Natural Deep Eutectic Solvents as a Key Metal Extractant for Fractionation in Speciation Analysis.

The research aimed to use natural deep eutectic solvents (NADES) as an extractant for fractionation of compounds of selected elements from young barley and to compare it with the fractionation of elements from certified element materials. The use of such a comparison made it possible to prove the possibility of extracting the same forms of elements (species) from different materials, which confirmed the option of using NADES as extractants in speciation analysis. The research was conducted with hyphenated techniques-separation by high-performance chromatography coupled to an isotope-specific detector, mass spectrometry (MS) with ionization in inductively coupled plasma (ICP)-which are widely used in speciation analysis. Natural deep eutectic solvents also help introduce Green Analytical Chemistry principles (GAC). According to the results of our studies, the use of different NADES permit the extraction of various metals from a single sample. Moreover, using other natural solvents of eutectic properties helps extract different species of a given metal.

Preliminary studies of the impact of food components on nutritional properties of nanoparticles.

Nowadays consumers have constantly exposed to nanoparticles (NPs) ingestion. Although the impact of NPs on the human has been studied by many authors, they did not consider the influence of food matrix components on bioaccessibility of NPs. This fact has encouraged us to investigate the influence of different food components on NPs. The investigation has been carried out to assess the influence of main food components on the MNPs (metallic nanoparticles) fate during the in vitro gastrointestinal simulation. The experiments have been carried out with the single-particle inductively coupled plasma mass spectrometry (SP-ICP-MS) as a tool for quantitative and qualitative analysis and the scanning transmission electron microscopy (STEM) as a means of qualitative analysis. The influence of various food components on NPs has been confirmed and it may be concluded that the matrix has an impact on the size and form of NPs. The presence of food components significantly changes the behaviour of NPs during simulated gastrointestinal digestion. Possible explanations of the influence of main nutrient groups, i.e. lipids, protein, salts, saccharides and vitamins on NPs have been proposed.

Application of Natural Deep Eutectic Solvents for the metal nanoparticles extraction from plant tissue.

The study aimed to use Natural Deep Eutectic Solvents (NADES) as an extractant of metal oxide NPs from plant material. The plant chosen for the study was radish after exposure, growing on media containing: copper(II) oxide, cerium(IV) oxide, and titanium(IV) oxide. The first step of the study was to investigate the influence of NADES on NPs. In the second step, selected NADES solvents were used as extractants of NPs present in radish after exposure. Single-particle Inductively Coupled Plasma Mass Spectrometry technique (SP-ICP-MS) was used to determine the number and size of NPs. As a result of the research, it was found that copper(II) oxide NPs, regardless of the solvent used, is not present in the extract. Only the ionic form of the element was present in the solution. Higher sized cerium(IV) oxide NPs were accumulated in the root, while smaller sized ones were found in radish leaves. The titanium(IV) oxide NPs were agglomerated and were present in a small amount in radish leaves, accumulating mainly in the root. Finally, it can be concluded that NADES allows the extraction of metal oxide NPs from the plant material.

To-Do and Not-To-Do in Model Studies of the Uptake, Fate and Metabolism of Metal-Containing Nanoparticles in Plants.

Due to the increasing release of metal-containing nanoparticles into the environment, the investigation of their interactions with plants has become a hot topic for many research fields. However, the obtention of reliable data requires a careful design of experimental model studies. The behavior of nanoparticles has to be comprehensively investigated; their stability in growth media, bioaccumulation and characterization of their physicochemical forms taken-up by plants, identification of the species created following their dissolution/oxidation, and finally, their localization within plant tissues. On the basis of their strong expertise, the authors present guidelines for studies of interactions between metal-containing nanoparticles and plants.

Analysis of cerium oxide and copper oxide nanoparticles bioaccessibility from radish using SP-ICP-MS.

BACKGROUND: The transformation of nanoparticles (NPs) internalized in plant tissues is the human digestive system that can provide a better understanding of the impact of NPs on the human system. The presented methodology was developed to study the bioaccessibility of cerium oxide (CeO2 ) and copper oxide (CuO) NPs from radish after the in vitro simulation of gastrointestinal digestion using single-particle inductively coupled plasma mass spectrometry (SP-ICP-MS). RESULTS: Radish plants were cultivated hydroponically in a growth medium containing: (i) CeO2 NPs and (ii) CuO NPs. Both cerium (Ce) and copper (Cu) were found in all organs of the radish plants after analysis by standalone ICP-MS. This confirms the bioaccumulation of CeO2 and CuO NPs and the translocation of their Ce and Cu to the aerial parts of the plant. Less Ce (4.095 µg g-1 ) has been detected in leaves than in roots (1.156 mg g-1 ) while Cu content in leaves was 5.245 µg g-1 and in roots was 10.41 µg g-1 . Analysis of the digestive extracts obtained after the in vitro simulation of gastro (pepsin) and gastrointestinal (pancreatin) digestion showed that Ce has easy access to human system at least by 73%. CONCLUSION: The size of CeO2 NPs in digestive extracts showed no significant changes. However, the results obtained for CuO NPs digestion were variable and suggested that CuO NPs dissolved during the digestion process. The CuO NPs were observed in roots after the gastrointestinal digestion concluding that CuO NPs recovered after the initial dissolution. © 2020 Society of Chemical Industry.

Uptake, translocation, size characterization and localization of cerium oxide nanoparticles in radish (Raphanus sativus L.).

Due to their unique physical and chemical properties, the production and use of cerium oxide nanoparticles (CeO2 NPs) in different areas, especially in automotive industry, is rapidly increasing, causing their presence in the environment. Released CeO2 NPs can undergo different transformations and interact with the soil and hence with plants, providing a potential pathway for human exposure and leading to serious concerns about their impact on the ecosystem and human organism. This study investigates the uptake, bioaccumulation, possible translocation and localization of CeO2 NPs in a model plant (Raphanus sativus L.), whose edible part is in direct contact with the soil where contamination is more likely to happen. The stability of CeO2 NPs in plant growth medium as well as after applying a standard enzymatic digestion procedure was tested by single particle ICP-MS (SP-ICP-MS) showing that CeO2 NPs can remain intact after enzymatic digestion; however, an agglomeration process was observed in the growth medium already after one day of cultivation. An enzymatic digestion method was next used in order to extract intact nanoparticles from the tissues of plants cultivated from the stage of seeds, followed by size characterization by SP-ICP-MS. The results obtained by SP-ICP-MS showed a narrower size distribution in the case of roots suggesting preferential uptake of smaller nanoparticles which led to the conclusion that plants do not take up the CeO2 NPs agglomerates present in the medium. However, nanoparticles at higher diameters were observed after analysis of leaves plus stems. Additionally, a small degree of dissolution was observed in the case of roots. Finally, after CeO2 NPs treatment of adult plants, the spatial distribution of intact CeO2 NPs in the radish roots was studied by laser ablation ICP-MS (LA-ICP-MS) and the ability of NPs to enter and be accumulated in root tissues was confirmed.

Analytical methodology for studying cellular uptake, processing and localization of gold nanoparticles.

Interactions of gold nanoparticles (AuNPs) with live cells are known to exert a great impact on their functions, including cell signalling, genomic, proteomic, and metabolomic processes. Modern analytical techniques applied to studying nanoparticle-cell interactions are to improve our understanding of the mode of action of AuNPs, which is essential for their approval in disease therapeutics. Such methods may vary depending on what step of particle internalization is in question, i.e., cellular uptake, intracellular transport (accompanying by changes in the chemical state), translocation to different cell compartments, interaction with relevant subcellular structures and localization. This review focuses on the implementation and critical assessment of advanced analytical methodologies to investigate the cellular processing of AuNPs. Also addressed is a sought-after issue of accounting in in-vitro studies for a chemical form in which the AuNPs enter the cell in vivo.

Application of CE-ICP-MS and CE-ESI-MS/MS for identification of Zn-binding ligands in Goji berries extracts.

The identification of groups of ligands binding metals is a crucial issue for the better understanding of their bioaccessibility. In the current study, we have intended an approach for identification of Zn-binding ligands based on using capillary electrophoresis combined with inductively coupled plasma mass spectrometry (CE-ICP-MS) and tandem electrospray ionization mass spectrometry (CE-ESI-MS/MS). The approach, which featured the use of the coupling of capillary electrophoresis with inductively coupled plasma mass spectrometry allows to separate and observe zinc ions present in complexes with respect to their size and charge and to identify nine compounds with zinc isotopic profile. CE-ICP-MS provides us with information about presence of zinc species and elemental information about zinc distribution. CE-ESI-MS/MS provide us with information about the most favorable Zn binding ligands: amino acids, flavonols, stilbenoids, fenolic acids and carotenoids. The presented work is the continuation of previous studies based on using LC-ESI-MS/MS, though, now we presented a new solutions with the possibility of changing detectors without changing the separation techniques, what is important without re-optimizing the method. The new presented method allows to identify the zinc-binding ligands in shorter time.

Speciation analysis and bioaccessibility evaluation of trace elements in goji berries (Lycium Barbarum, L.).

Goji berries (Lycium Barbarum, L.) are known for their nutritional potential as a great source of trace metals (e.g., copper, zinc and manganese) which are present in the form of highly bioaccessible compounds. In order to assess the bioaccessibility of trace elements and to identify compounds responsible for better bioaccessibility of copper and zinc, an in vitro simulation of gastrointestinal digestion was used in this study. The total content of trace metals was evaluated using sample digestion followed by inductively coupled plasma mass spectrometry. Bioaccessibility of trace elements was estimated by size exclusion chromatography coupled to inductively coupled plasma mass spectrometry. These analytical methods were used to analyse samples of goji berries to determine the highest amount of elements. For total trace metal content in goji berries, Zn had the highest level of the three studied (10.6µgg-1), while the total content of manganese and copper was 9.9µgg-1 and 6.1µgg-1, respectively. Additionally, the analysed metals were found to be highly bioaccessible to the human body (about 56% for Mn, 72% for Cu and 64% for Zn in the gastric extract and approximately 35% for Mn, 23% for Cu and 31% for Zn in the case of gastrointestinal extract). To obtain information about metal complexes present in goji berries, extraction treatment using different solutions (ionic liquid, HEPES, SDS, Tris-HCl, ammonium acetate, water) was performed. Enzymatic treatment using pectinase and hemicellulase was also checked. Extracts of berries were analysed by SEC-ICP-MS and µHPLC-ESI-MS/MS techniques. The ionic liquid and pectinase extraction helped efficiently extract copper (seven compounds) and zinc (four compounds) complexes. Compounds identified in goji berries are most likely to be responsible for better bioaccessibility of those elements to the human organism.

In vitro digestion method for estimation of copper bioaccessibility in Açaí berry.

ABSTRACT: Copper is an essential trace element for humans and its deficiency can lead to numerous diseases. A lot of mineral supplements are available to increase intake of copper. Unfortunately, only a part of the total concentration of elements is available for human body. Thus, the aim of the study was to determine bioaccessibility of copper in Açai berry, known as a "superfood" because of its antioxidant qualities. An analytical methodology was based on size exclusion chromatography (SEC) coupled to a mass spectrometer with inductively coupled plasma (ICP MS) and on capillary liquid chromatography coupled to tandem mass spectrometer with electrospray ionization (µ-HPLC-ESI MS/MS). To extract various copper compounds, berries were treated with the following buffers: ammonium acetate, Tris-HCl, and sodium dodecyl sulfate (SDS). The best extraction efficiency of copper was obtained for SDS extract (88 %), while results obtained for Tris-HCl and ammonium acetate were very similar (47 and 48 %, respectively). After SEC-ICP-MS analysis, main signal was obtained for all extracts in the region of molecular mass about 17 kDa. A two-step model simulated gastric (pepsin) and gastrointestinal (pancreatin) digestion was used to obtain the knowledge about copper bioaccessibility. Copper compounds present in Açai berry were found to be highly bioaccessible. The structures of five copper complexes with amino acids such as aspartic acid, tyrosine, phenylalanine, were proposed after µ-HPLC-ESI MS/MS analysis. Obtained results show that copper in enzymatic extracts is bound by amino acids and peptides what leads to better bioavailability of copper for human body.

Ionic liquids as a key medium for efficient extraction of copper complexes from chia seeds (Salvia hispanica L.).

Due to insufficient information, the aim of study was to concern on the optimization of extraction procedure of selected metal complexes with flavonoids from chia seeds. Evaluation of the amount of elements in compound, not only their total concentration content, is highly important due to the fact, that only a part from total content of metal is absorbed by human body. At the beginning the total amount of elements in chia seeds was established as 14.51±0.42 µg g(-1) for copper, 57.44±1.23 µg g(-1) for manganese, 81.12±1.89 µg g(-1) for zinc and 0.35±0.13 µg g(-1) for cobalt. After the most suitable solvent was established, effects of several parameters on the efficiency of metal extraction were studied. Solvent concentration, solid-solvent ratio, extraction method, extraction time and temperature have been investigated as independent variables. The optimal extraction conditions included vortexing during 20 min in 50°C, using an ionic liquid (1-butyl-3-methylimidazolium bromide) as an extractant, with solid-solvent ratio of 1:20. The determination of total and extractable amount of metals in chia seeds was carried out by standalone ICP MS. In addition, a complementary analysis of extracted metal complexes was performed using SEC-ICP MS method. It was confirmed that the ionic liquid is able to extract different copper complexes in comparison with commonly used solvents. The study indicated that extraction by using an ionic liquid has been successfully applied for determination of metals and metal complexes in chia seeds.

Operationally defined species characterization and bioaccessibility evaluation of cobalt, copper and selenium in Cape gooseberry (Physalis Peruviana L.) by SEC-ICP MS.

Physalis peruviana could attract great interest because of its nutritional and industrial properties. It is an excellent source of vitamins, minerals, essential fatty acids and carotenoids. Physalis Peruviana is also known to have a positive impact on human health. Unfortunately, still little is known about trace elements present in Physalis Peruviana and their forms available for the human body. Thus, the aim of this study was to estimate bioaccessibility and characterization of species of cobalt, copper and selenium in Physalis Peruviana fruits. Total and extractable contents of elements were determined by mass spectrometer with inductively coupled plasma (ICP MS). In order to separate the different types of metal complexes Physalis peruviana fruits were treated with the following solvents: Tris-HCl (pH 7.4), sodium dodecyl sulfate (SDS) (pH 7.4) and ammonium acetate (pH 5.5). The best efficiency of extraction of: cobalt was obtained for ammonium acetate (56%) and Tris-HCl (60%); for copper was obtained for SDS (66%), for selenium the best extraction efficiency was obtained after extraction with SDS (48%). To obtain information about bioaccessibility of investigated elements, enzymatic extraction based on in vitro simulation of gastric (pepsin) and intestinal (pancreatin) digestion was performed. For copper and selenium the simulation of gastric digestion leads to the extraction yield above 90%, while both steps of digestion method were necessary to obtain satisfactory extraction yield in the case of cobalt. Size exclusion chromatography (SEC) coupled to on-line ICP MS detection was used to investigate collected metal species. The main fraction of metal compounds was found in the 17 kDa region. Cobalt and copper create complexes mostly with compounds extracted by means of ammonium acetate and SDS, respectively. Cobalt, copper and selenium were found to be highly bioaccessible from Physalis Peruviana. Investigation of available standards of cobalt and selenium allows confirming the presence of vitamin B12 and probably selenomethionine in the fraction bioaccessible by human body (obtained during enzymatic extraction). It should be noted that the presence of small seleno-compounds in Cape gooseberry was performed for the first time. The results show that the combination of SEC and ICP MS could provide a simple method for separating of soluble element species.

Comparison of copper and zinc in vitro bioaccessibility from cyanobacteria rich in proteins and a synthetic supplement containing gluconate complexes: LC-MS mapping of bioaccessible copper complexes.

An analytical procedure was proposed to estimate bioaccessibility of copper and zinc in Spirulina Pacifica tablets with respect to that of copper and zinc in gluconate complexes. Spirulina is the common name for diet supplements produced primarily from two species of cyanobacteria, namely Arthrospira platensis and Arthrospira maxima. Spirulina tablets are an excellent source of proteins, vitamins and minerals. To obtain information about the bioavailability of these elements, an in vitro bioaccessibility test was performed by application of a two-step protocol which simulated the gastric (pepsin) and intestinal (pancreatin) digestion. The species obtained were investigated by size exclusion chromatography on a chromatograph coupled to a mass spectrometer with inductively coupled plasma (SEC-ICP-MS) and an on-capillary liquid chromatograph coupled to an electrospray mass spectrometer (µ-HPLC-ESI-MS). Both copper and zinc were found to be highly bioaccessible in Spirulina tablets (90-111%) and those containing gluconate complexes (103% for Cu and 62% for Zn). In Spirulina tablets, copper was found to form two types of complex: (1) polar ones with glycine and aspartic acid and (2) more hydrophobic ones containing amino acids with cyclic hydrocarbons (phenylalanine, histidine, proline and tyrosine). Zinc and copper were also proved to form complexes during the digestion process with products of pepsin digestion, but the stability of these complexes is lower than that of the complexes formed in Spirulina. The results proving the involvement of proteins in the enhancement of copper and zinc bioaccessibility will be useful for the design of new copper and zinc supplements.