Pressurized liquid extraction assisted by high-intensity ultrasound to obtain hesperidin from lime waste: Integration of in-line purification and on-line chromatographic analysis.

Hesperidin is a phenolic compound usually found in citrus fruits, which is known for its anti-inflammatory and antioxidant properties. This bioactive compound has already been used to formulate medications to treat chronic venous insufficiency. In this work, through a system which allows the in-line coupling of the pressurized liquid extraction (PLE) and high-intensity ultrasound (HIUS) with solid phase extraction (SPE), and analysis by high-performance liquid chromatography with UV-Vis detector (HPLC-UV) in on-line mode, a method was developed to obtain, separate, and quantify hesperidin from the industrial waste of lime. An eco-friendly approach with water and ethanol as extraction solvents was used. Parameters such as temperature (80, 100, and 120 °C) and HIUS power (0, 200, and 400 W) were evaluated regarding hesperidin yield. In this context, the higher hesperidin yield (18.25 ± 1.52 mg/g) was achieved using water at a subcritical state (120 °C and 15 MPa). The adsorbent SepraTM C-18-E isolated hesperidin from the other extracted compounds employing 50% ethanol in the SPE elution. The possibility ofon-lineanalysis coupling a high-performance liquid chromatograph to an ultraviolet detector (HPLC-UV) system was studied and shown to be a feasible approach for developing integrated technologies. Conventional extractions and their antioxidant capacities were evaluated, highlighting the advantages of the HIUS-PLE-SPE extractive method. Furthermore, the on-linechromatographic analysis showed the potential of the HIUS-PLE-SPE- HPLC-UV system to quantify the extracted compounds in real time.

Balancing the benefits of vaccination: An envy-free strategy.

The Covid-19 pandemic revealed the difficulties of vaccinating a population under the circumstances marked by urgency and limited availability of doses while balancing benefits associated with distinct guidelines satisfying specific ethical criteria. We offer a vaccination strategy that may be useful in this regard. It relies on the mathematical concept of envy-freeness. We consider finding balance by allocating the resource among individuals that seem heterogeneous concerning the direct and indirect benefits of vaccination, depending on age. The proposed strategy adapts a constructive approach in the literature based on Sperner's Lemma to point out an approximate division of doses guaranteeing that both benefits are optimized each time a batch becomes available. Applications using data about population age distributions from diverse countries suggest that, among other features, this strategy maintains the desired balance, throughout the entire vaccination period. We discuss complementary aspects of the method in the context of epidemiological models of age-stratified Susceptible - Infected - Recovered (SIR) type.

Simultaneous extraction, separation, and analysis of 5-caffeoylquinic acid and caffeine from coffee co-product by PLE-SPE × HPLC-PDA two-dimensional system.

This study proposed an integrated and automated procedure to extract, separate, and quantify bioactive compounds from a coffee co-product by pressurized liquid extraction (PLE) coupled inline with solid phase extraction (SPE) and online with HPLC-PDA (PLE-SPE × HPLC-PDA). The efficiency of the two-dimensional system in performing real-time analysis was verified by comparing HPLC-PDA results acquired by the system (online) and carried out after the extract fraction collection (offline). Different flow rates (1.5 mL/min for 336 min, 2 mL/min for 246.4 min, and 2.5 mL/min for 201.6 min) were evaluated to optimize the extraction, separation, and analysis method by PLE-SPE × HPLC-PDA. Subcritical water at 125 °C and 15 min of static time allowed the highest extraction yields of caffeine and 5-caffeoylquinic acid (5-CQA). Caffeine was retained during the aqueous extraction in the SPE adsorbent and eluted from the column by exchanging the solvent for a hydroethanolic mixture. Thus, caffeine was separated from 5-CQA and other phenolic compounds, producing extracts with different compositions. The solvent flow rate did not have a significant effect (p-value ≥ 0.05) on the extraction, separation, and analysis (by online and offline methods) of 5-CQA. However, the online quantification of retained compounds in the SPE (i.e., caffeine) can underestimate concentration compared to offline analysis. Nevertheless, the results suggest that coupling of advanced techniques can be used to efficiently extract, separate, and analyze fractions of phenolic compounds, supplying an integrated method to produce high-added value ingredients for several applications.

Fast and green universal method to analyze and quantify anthocyanins in natural products by UPLC-PDA.

This work developed a universal UPLC-PDA method based on safe reagents to analyze anthocyanins from different foods. Nine foods were studied by the developed chromatographic method, which was constructed using a solid core C18 column and a binary mobile phase composed of (A) water (0.25 molcitric acid.Lsolvent-1), and (B) ethanol. A total running time of 6 min was obtained, the faster comprehensive method for anthocyanins analysis. Mass spectrometry analysis was employed to identify a comprehensive set of 53 anthocyanins comprising glycosylated and acylated cyanidin, pelargonidin, malvidin, peonidin, petunidin, and delphinidin derivatives. Cyanidin-3-O-glucoside (m/z+ 449) and cyanidin-3-O-rutinoside (m/z+ 595) were used as standards to validate the accuracy of the developed method. The analytical parameters were evaluated, including intra-day and inter-day precision, robustness, repeatability, retention factor (k), resolution, and peak symmetry factor. The current method demonstrated excellent chromatographic resolution, making it a powerful tool for analyzing anthocyanins pigments.

High-intensity ultrasound-based process strategies for obtaining edible sunflower (Helianthus annuus L.) flour with low-phenolic and high-protein content.

The sunflower Helianthus annuus L. represents the 4th largest oilseed cultivated area worldwide. Its balanced amino acid content and low content of antinutrient factors give sunflower protein a good nutritional value. However, it is underexploited as a supplement to human nutrition due to the high content of phenolic compounds that reduce the sensory quality of the product. Thus, this study aimed at obtaining a high protein and low phenolic compound sunflower flour for use in the food industry by designing separation processes with high intensity ultrasound technology. First, sunflower meal, a residue of cold-press oil extraction processing, was defatted using supercritical CO2 technology. Subsequently, sunflower meal was subjected to different conditions for ultrasound-assisted extraction of phenolic compounds. The effects of solvent composition (water: ethanol) and pH (4 to 12) were investigated using different acoustic energies and continuous and pulsed process approaches. The employed process strategies reduced the oil content of sunflower meal by up to 90% and reduced 83% of the phenolic content. Furthermore, the protein content of sunflower flour was increased up to approximately 72% with respect to sunflower meal. The acoustic cavitation-based processes using the optimized solvent composition were efficient in breaking down the cellular structure of the plant matrix and facilitated the separation of proteins and phenolic compounds, while preserving the functional groups of the product. Therefore, a new ingredient with high protein content and potential application for human food was obtained from the residue of sunflower oil processing using green technologies.

Trace Elements in Soy-Based Beverages: A Comprehensive Study of Total Content and In Vitro Bioaccessibility.

Soy-based beverages are one of the most consumed plant-based beverages, which have been used as a substitute for dairy products. Soy is a source of several nutrients (vitamins, minerals, and phenolic compounds, etc.) and its consumption is usually associated with several benefits, such as the prevention of cardiovascular diseases, cancer, and osteoporosis. However, non-essential trace elements can be found in these beverages. Thus, a comprehensive study concerning trace elements Al, As, Cd, Co, Cr, Cu, Fe, Li, Mn, Ni, Pb, Sb, Se, Sn, Sr, and Zn in soy-based beverages was proposed. In vitro digestion allowed to simulate the gastrointestinal juice (bioaccessibility) and the Caco-2 cells culture model was applied for the bioavailability assay. Trace elements measures were performed by inductively coupled plasma optical emission spectrometry (ICP OES). Multivariate analysis classified soy-based beverages according to their soy source (isolate protein, hydrosoluble extract, and beans); Al, Cu, Fe, Mn, Sr, Se, and Zn bioaccessible fractions corresponded to approximately 40%-80% of their total content, and soy-based beverages were found to be a good Fe, Se, and Zn source. However, our results showed risk exposure assessment from daily consumption of one glass of soy-based beverage can contribute to 3.5% and 0.9% of Al Provisional Tolerable Weekly Intake (PTWI) for children and adults, respectively.

Simultaneous extraction and analysis of apple pomace by gradient pressurized liquid extraction coupled in-line with solid-phase extraction and on-line with HPLC.

Due to the complex characteristics and variable composition of apple pomace, sample preparation for chromatographic analysis is a great challenge. To solve this problem, we proposed using a solvent gradient using Pressurized Liquid Extraction (PLE), where the solvent gradually changes from water to ethanol during the extraction. Different dynamic gradients, static time, and temperatures were evaluated and showed relevant effects on the yields of target analytes. It was possible to improve extraction yields of compounds with different characteristics using the extraction solvent gradient. By coupling solid-phase extraction in-line, it was possible to separate compounds into fractions, where furfural, HMF, and chlorogenic acid gradually eluted from the adsorbent. At the same time, flavonoids were retained and eluted in the later fractions. On-line analysis by HPLC provided real-time information about the process and permitted the creation of a 3D chromatogram of the sample.

Ultra-high-performance liquid chromatography using a fused-core particle column for fast analysis of propolis phenolic compounds.

Propolis is a bee product with a complex chemical composition formed by several species from different geographical origins. The complex propolis composition requires an accurate and reproducible characterization of samples to standardize the quality of the material sold to consumers. This work developed an ultra-high-performance liquid chromatography with a photodiode array detector method to analyze propolis phenolic compounds based on the two key propolis biomarkers, Artepillin C and p-Coumaric acid. This choice was made due to the complexity of the sample with the presence of several compounds. The optimized method was hyphenated with mass spectrometry detection allowing the detection of 23 different compounds. A step-by-step strategy was used to optimize temperature, flow rate, mobile phase composition, and re-equilibration time. Reverse-phase separation was achieved with a C18 fused-core column packed with the commercially available smallest particles (1.3 nm). Using a fused-core column with ultra-high-performance liquid chromatography allows highly efficient, sensitive, accurate, and reproducible determination of compounds extracted from propolis with an outstanding sample throughput and resolution. Optimized conditions permitted the separation of the compounds in 5.50 min with a total analysis time (sample-to-sample) of 6.50 min.

Standardization proposal to quality control of propolis extracts commercialized in Brazil : A fingerprinting methodology using a UHPLC-PDA-MS/MS approach.

Propolis is a rich source of known and largely explored bioactive compounds with many pharmacological properties. It is used in several commercialized products, such as propolis-enriched honey, candies, mouth and throat sprays, soaps, toothpaste, and skin creams. However, the great diversity of propolis products and different types make the standardization of realistic quality control procedures challenging. Moreover, the extraction of propolis bioactive compounds depends on the technique and the solvent used. In Brazil, the Ministry of Agriculture, Livestock, and Supply (MAPA) set standards to establish commercialized propolis extracts' identity and quality. In addition, according to legislation, propolis extracts must present the main classes of phenols at 200 and 400 nm on the UV spectrum. Still, it is not specified which analysis method should be used to guarantee feasible quality control of the commercialized samples. For this, we proposed a new fast UHPLC-PDA-MS/MS method for analysis and quantification of propolis phenolic compounds. Moreover, we hypothesize that there is no efficient monitoring regarding the quality of the propolis extracts sold in Brazilian stores. Therefore, the present study aimed to perform quality control of 17 Brazilian propolis extracts produced in the Southeast region (green or brown - the most representative samples). The dry extract content (% g/mL), oxidation index (seconds), total flavonoids, and phenolics (% m/m) of each sample were compared with legislation. We conclude that using the UHPLC-PDA method and the investigation that allowed the comparison with the current legislation efficiently practical problems in the commercialization of propolis extracts. However, of the 17 analyzed samples, 6 did not meet the desired the recognized standards, denoting a lack of supervision and efficient quality control, which highlights a dangerous situation regarding the commercialization of this critical product used in several industrial fields, mainly in the food and pharmaceutical sector.

Insights on the Extraction and Analysis of Phenolic Compounds from Citrus Fruits: Green Perspectives and Current Status.

Citrus fruits (CF) are highly consumed worldwide, fresh, processed, or prepared as juices and pies. To illustrate the high economic importance of CF, the global production of these commodities in 2021 was around 98 million tons. CF's composition is considered an excellent source of phenolic compounds (PC) as they have a large amount and variety. Since ancient times, PC has been highlighted to promote several benefits related to oxidative stress disorders, such as chronic diseases and cancer. Recent studies suggest that consuming citrus fruits can prevent some of these diseases. However, due to the complexity of citrus matrices, extracting compounds of interest from these types of samples, and identifying and quantifying them effectively, is not a simple task. In this context, several extractive and analytical proposals have been used. This review discusses current research involving CF, focusing mainly on PC extraction and analysis methods, regarding advantages and disadvantages from the perspective of Green Chemistry.

Integration of pressurized liquid extraction and in-line solid-phase extraction to simultaneously extract and concentrate phenolic compounds from lemon peel (Citrus limon L.).

This work aimed to develop an integrated method to extract and fractionate phenolic compounds from lemon (Citrus limon L.) peel by in-line coupling pressurized liquid extraction and solid-phase extraction (PLE-SPE). The effect of the adsorbent used in the SPE (Sepra™ C18-E, Sepra™ NH2, and PoraPak Rxn), the combination of organic extraction-elution solvents (water-ethanol and water-ethyl lactate), extraction temperature (40-80 °C), and extraction water pH (4.0, 6.0, and 7.0) were the investigated variables. The highest yield and separation degree were observed using Sepra™ C18-E and the water-ethanol combination as the extraction solvent-eluent. Higher temperatures led to higher yields but negatively affected the retention of less polar compounds, hesperidin, and narirutin during the extraction step. The lower pH improved the yield of most evaluated compounds; however, it did not improve the adsorbent retention at high temperatures. Thus, the developed PLE-SPE method resulted in higher extraction yields from lemon peel, especially total less polar compounds (20.2100 ± 0,0050 mg/g) and hesperidin (12.8120 ± 0.0006 mg/g) and allowed the separation of polar compounds and less polar compounds in distinct extract fractions. Besides, PLE-SPE resulted in higher yields compared to other extraction methods. The integrated approach allowed obtaining extract fractions with different chemical composition through an environmentally friendly procedure. The research outcomes may be helpful for natural products chemistry, and industrial processes.

Extraction of potential bioactive compounds from industrial Tahiti lime (Citrus latifólia Tan.) by-product using pressurized liquids and ultrasound-assisted extraction.

This work evaluated two emerging techniques in extracting phenolic compounds from Tahiti lime pomace - pressurized liquid extraction (PLE) and ultrasound-assisted extraction (UAE). PLE was performed at different temperatures (60 - 110 °C) and times (5 - 40 min), and UAE was carried out varying ultrasound power (160 - 792 W), time (2 - 10 min), and solvent to feed mass ratio (20 - 40 kg solvent/kg dried pomace). Both used ethanol and water (3:1, wt.) as the solvent. The effects of these variables were evaluated on global extraction yield, polyphenols, hesperidin, narirutin yields, and antioxidant capacity. PLE was strongly affected by temperature and extraction time, and the highest temperature (110 °C) provided the best results for global yield, total phenolic, and ORAC, except for hesperidin and narirutin, which were not significative affected by temperature. UAE revealed a weak dependency on power, S/F, and time; however, the lowest power level significantly increased the yields compared to no power application. Thus, P = 480 W, t = 6 min, and S/F = 30 was chosen as the best condition in the UAE in terms of overall extraction yield, total phenolics, specific phenolics, antioxidant capacities, and solvent and energy expenditures. UAE mechanisms were investigated by comparing with heated and stirred maceration, and scanning electron microscopy suggested that total phenolic yield was influenced by mechanisms that only ultrasound can provide. Micrographics confirmed the cavitation effect on Tahiti lime pomace particles' surface. To sum up, PLE resulted in the highest yields and antioxidant capacity, followed by UAE.

Formulations with microencapsulated Fe-peptides improve in vitro bioaccessibility and bioavailability.

The bioaccessibility and the bioavailability of iron complexed to peptides (active) in microparticles forms contained in dry beverages formulations were evaluated. The peptide-iron complexes microparticles were obtained by spray drying and added in three dry formulations (tangerine, strawberry, and chocolate flavors). The peptides isolated by iron ion affinity (IMAC-Fe III) had their biological activity predicted by BIOPEP® database and were evaluated by molecular coupling. The bioaccessibility was evaluated by solubility and dialysability and the bioavalability was assessed by Caco-2 cellular model. The proportion 10:1 of peptide-iron complexes presented higher rates of bioaccessibility (49%) and bioavailability (56%). The microparticle with peptide-iron complex showed greater solubility after digestion (39.1%), bioaccessibility (19.8%), and bioavailability (34.8%) than the ferrous sulfate salt (control) for the three assays (10.2%; 12.9%; 9.7%, respectively). Tangerine and strawberry formulations contributed to the iron absorption according to the results of bioaccessibility (36.2%, 30.0% respectively) and bioavailability (80.5%, 84.1%, respectively). The results showed that iron peptide complexation and microencapsulation process improve the bioaccessibility and bioavailability when incorporated into formulations.

Comprehensive analysis of phenolics compounds in citrus fruits peels by UPLC-PDA and UPLC-Q/TOF MS using a fused-core column.

In this work, a method based on ultra-high-performance liquid chromatography with a photodiode array detector (UPLC-PDA) was developed to comprehensively analyze phenolic compounds in peels of lime (Citrus × latifolia), lemon (Citrus limon), and rangpur lime (Citrus × limonia). The reverse-phase separation was achieved with a C18 fused-core column packed with the smallest particles commercially available (1.3 um). The method was successfully coupled with high-resolution mass spectrometry (HRMS), allowing the detection of 24 phenolic compounds and five limonoids in several other citrus peels species: key lime, orange and sweet orange, tangerine, and tangerine ponkan, proving the suitability for comprehensive analysis in citrus peel matrices. Additionally, the developed method was validated according to the Food and drug administration (FDA) and National Institute of Metrology Quality and Technology (INMETRO) criteria, demonstrating specificity, linearity, accuracy, and precision according to these guidelines. System suitability parameters such as resolution, tailoring, plate count were also verified.

An integrated model to study varietal diversity in traditional agroecosystems.

Agricultural diversity is one of the bases of traditional agroecosystems, having great environmental and cultural importance. The current loss of agricultural diversity is causing serious concern, mainly because of its essential role in supporting global and local food security. Stopping this loss requires a better understanding of how diversity is managed locally and what mechanisms sustain agricultural diversity. Here we propose a generalist agent-based model that couples biological, cultural, and social dynamics to obtain varietal diversity as an emergent phenomenon at the community level. With a mechanistic approach, we explore how four of the model dynamics can shape systems diversity. To validate the model, we performed a bibliographic review on Manihot esculenta and Zea mays case studies. The model yielded compatible results for manioc and maize varietal richness at both community and household levels.

Recent advances and trends in extraction techniques to recover polyphenols compounds from apple by-products.

Apple is one of the most consumed fruits worldwide and has recognized nutritional properties. Besides being consumed fresh, it is the raw material for several food products, whose production chain generates a considerable amount of by-products that currently have an underestimated use. These by-products are a rich source of chemical compounds with several potential applications. Therefore, new ambitious platforms focused on reusing are needed, targeting a process chain that achieves well-defined products and mitigates waste generation. This review covers an essential part of the apple by-products reuse chain. The apple composition regarding phenolic compounds subclasses is addressed and related to biological activities. The extraction processes to recover apple biocompounds have been revised, and an up-to-date overview of the scientific literature on conventional and emerging extraction techniques adopted over the past decade is reported. Finally, gaps and future trends related to the management of apple by-products are critically presented.

Comprehensive analysis of phenolic compounds from natural products: Integrating sample preparation and analysis.

The comprehensive analysis of phenolic compounds from natural products comprises critical steps, including quantitative extraction, extract preparation, and chromatographic procedure. Performing these steps off-line requires a long time to obtain results, besides being laborious and more error-prone. This work discusses the concept and presents the details of assembling and validating a new system to comprehensively analyze phenolic compounds in natural products. The system is based on a bidimensional separation through the combination of pressurized liquid extraction with in-line solid-phase extraction coupled online with HPLC-PDA. The system proved to be able to perform a bidimensional separation to characterize the sample and ensure quantitative extraction of all detected components using the most appropriate extraction solvent gradient depending on the raw sample analyzed. The 1st dimension separation is achieved by PLE-SPE with a solvent gradient and differential interactions of extracted compounds with the adsorbent. The 2nd dimension presents the HPLC-PDA separation. The extraction/separation process can be monitored in real-time, and kinetic extraction curves for individual compounds can also be obtained to ensure quantitative extraction. Thus, the 2D PLE-SPE × HPLC-PDA may provide fast and precise comprehensive analyses of a large plethora of phenolic compounds, finding relevant applications in the chemical, food, pharmaceutical, and agricultural fields.

Simultaneous extraction and separation of compounds from mate (Ilex paraguariensis) leaves by pressurized liquid extraction coupled with solid-phase extraction and in-line UV detection.

The in-line coupling of the pressurized liquid extraction with a solid-phase adsorbent and a UV-Vis detector for the simultaneous extraction and separation of bioactive compounds from yerba mate (PLE-SPE-UV) was carried out in two stages. In the first stage, water was used as a solvent, while in the second stage, ethanol was used. For the optimization of the method, different adsorbents (Sepra C18-E, Isolute C18-EC, and Strata-X C18), temperatures (40-80 °C), solvent flow-rate (1-3 mL/min), and pH (4.0 and 8.0) were evaluated. By using a UV-Vis detector on-line, it is possible to monitor the process in real-time. The developed method allowed obtaining similar or higher recoveries of all the compounds classes than other methods, such as ultrasound-assisted extraction, stirring, maceration, and pressurized liquid extraction alone, in addition to separating them into fractions. The developed method could be used as sample preparation for the analysis of different compounds classes from mate.

Trace elements in ready-to-drink ice tea: Total content, in vitro bioaccessibility and risk assessment.

Tea is one of the most consumed non-alcoholic beverages in world and it has been frequently associated to health benefits. Besides its nutrient composition, non-essential trace elements associated with toxic effects may also be present. Ever since food components undergo biotransformation process along gastrointestinal tract after ingestion, it is important to evaluate both total and bioavailable content of trace elements. Therefore, this study aimed to provide comprehensive data concerning the influence of the in vitro digestion on sixteen trace elements present in ready-to-drink ice tea (black, green, mate and white tea). Essential minerals (Co, Cr, Cu, Fe, Mn, Se and Zn) and inorganic contaminants (Al, As, Cd, Li, Ni, Pb, Sb, Sn and Sr) contents were determined by ICP OES after microwave acid digestion. Bioaccessibility evaluation was carried out by simulating the gastric (pepsin) and intestinal juice (pancreatin and bile salts) and bioavailability used Caco-2 cells culture as an intestinal epithelial model. Moreover, tannins were evaluated by UV-VIS spectroscopy. Multivariate analysis allowed classifying ice tea samples in three groups, based on their trace elements profile. Al, Cu, Sr, Mn and Zn bioaccessible fractions corresponded to, approximately, 40-60% of their total content. For Mn, bioaccessibility and bioavailability presented the same pattern (green ice tea > black ice tea > mate ice tea) whilst Sr bioavailability in green tea were 50% higher than in black tea samples.

Trace mineral composition of human breast milk from Brazilian mothers.

BACKGROUND: Human milk is a dynamic food and some important differences in composition can be found between the milk from preterm and terms infants. Additionally, in some situations, the mother's own milk is unavailable and the use of milk from human milk banks is considered as the most appropriate substitute. In this way, concentrations of trace elements (Ba, Cu, Fe, Mn, Mo, Se, Sr, and Zn) were determined in human milk, considering the differences about preterm and term human milk and its processing in a human milk bank. METHODS: A total of 156 samples were analyzed, which were divided in three groups: samples collected at the hospital at bedside (BS, 60 samples) from mothers of preterm infants and samples from mothers of term infants collected in a human milk bank without pasteurization (WP, 49 samples) and pasteurized by the Holder procedure (P, 47 samples). The analyzes were conducted by inductively coupled plasma mass spectrometry (ICP-MS) after the treatment of the samples with acid mineralization assisted by microwave radiation. RESULTS: Concentrations varied in a range of 0.6-88.2 µg/L for Ba, 78.6-954.5 µg/L for Cu, 24.2-5229.2 µg/L for Fe, 0.4-42.6 µg/L for Mn, 0.1-39.1 µg/L for Mo, 2.5-70.6 µg/L for Se, 8.9-187.5 µg/L for Sr and 76.3-17727.2 µg/L for Zn. Significant differences (p < 0.05) were found between preterm (BS) and term human milk (WP and P) for Ba, Cu, Mo, Se, and Zn, whereas the processing of the donated milk by Holder pasteurization did not influence the concentration of the studied trace elements. The milk of term infants does not attend the recommended daily intake (RDI) of Zn and for preterm infants the RDI of Fe and Mn is not achieved. CONCLUSIONS: The higher concentrations of Cu, Mo, Se and Zn observed in milk from mothers of preterm infants indicate that the milk to be offered for these high-risk neonates in neonatal intensive care units should contain higher levels of these trace elements. Besides, considering the RDI, the milk of term infants should be fortified with Zn, whereas the milk of preterm infants should be fortified with Fe.