Thin layer chromatography bioautographic assays facilitate the acquisition of activity-profile chromatograms and assist in pinpointing active constituents within complex mixtures by observing the inhibition halos they produce. Peroxidase is an enzyme implicated in the browning of different fresh cut vegetables and in several diseases. A peroxidase bioautographic assay was developed, based on enzyme agarose immobilization and the 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt/radical cation (ABTS/ABTS·+) reporter system. Peroxidase was purified from potatoes with the aim to detect specific inhibitors. To reduce false positives, a non-enzymatic assay was also employed. The best results are obtained when a solution containing agarose, ABTS, hydrogen peroxide, and peroxidase in phosphate buffer is poured over the TLC plate (final concentrations: 0.031 mmoles/cm2, 0.239 µmoles/cm2, and 84.04 U/cm2) and incubated for 70 min. Limit of detection and quantification for quercetin is 0.16 µg and 0.54 µg, respectively. The developed system is able to detect quercetin in a Solidago chilensis Meyen extract and a peroxidase inhibitor in a Cichorium intybus L. extract. Therefore, the assay can detect inhibitory constituents in complex mixtures and differentiate between peroxidase inhibitors and ABTS·+ radical scavengers before any preparative fractionation, helping to take early operational decisions that can save time and resources.

During the last decades, extrusion has been raised as an innovative technique for the development of novel food products. Its versatility allows the combination of different raw materials, which facilitates the generation of new products with improved nutritional profiles, enhanced sensory or functional characteristics, or better physicochemical properties at low costs. Extrusion can also be considered a valuable tool for food waste utilization, thus helping to close the loop of the food value chain. Due to its unique characteristics of blending, cooking, and forming in the same step by a combination of mechanical energy and thermal input, extrusion allows the incorporation of a wide variety of undervalued grains or by-products from other food production lines, thus promoting a circular economy and contributing to the accomplishment of the Sustainable Development Goals established by the United Nations. Nevertheless, to achieve desired characteristics, it is necessary to consider the thermo-mechanical transformations undergone in the process by the main components of the selected ingredients. In this chapter, we present general tips for the management of the different extrusion processing conditions, the inclusion of various ingredients of interest, and their effects on the final products– characteristics. Finally, we review the recent advances in food extrudates developments.

The use of natural antioxidant extracts in food processing is a growing trend, aligning with increasing ecological awareness and meeting consumer demands for safe, high-quality food products. Plants play a significant role in human health owing, in part, to their antioxidant properties. Analyzing the antioxidant properties of complex natural extracts requires simple, cost-effective tools. The cupric reducing antioxidant capacity (CUPRAC) method is a reference for evaluating plant extract antioxidant capacity. A new CUPRAC thin-layer chromatography (TLC) assay, involving agarose immobilized reagents, was developed and applied to investigate the antioxidant properties of Solanum sisymbriifolium Lam., Sinapis arvensis L., and Cichorium intybus L. The best results were obtained by pouring a solution containing agarose, copper(II) chloride, 2,2-azino-bis[3-ethylbenzothiazoline-6-sulfonic acid] (ABTS), and neocuproine in ammonium acetate buffer over a TLC plate at 40 °C (with final concentrations of 0.485 µmoles/cm2 copper(II) chloride and 0.368 µmoles/cm2 neocuproine) and incubating for 15 min. The limits of detection and quantification for Trolox® were 0.09 and 0.30 µg, respectively.

Summary Corn‐based snacks were prepared with the addition of 25% of millet, sorghum, quinoa, and canary seed flours, which are scarcely used grains for human food and can grow in low‐fertility soils. Guggenheim‐Anderson‐de Boer (GAB) and D'arcy & Watt (GDW) sorption models provided information regarding water‐solids interactions and microstructural arrangements. After storage for 2, 4, and 6 months, several physicochemical properties of snacks were measured and compared with freshly prepared products and raw blends. Addition of quinoa and canary seed flours doubled phenolics content and antioxidant properties. Lipids oxidation kinetics was associated with water sorption, antioxidant properties, and carotenoids and phenolics stability. Starch gelatinisation and protein transformations were assessed through FTIR. Protein denaturation after thermal treatment reduced intermolecular associations with carotenoids and phenolic compounds, which enhanced their functionality as antioxidants. Addition of quinoa and canary seed flours showed promising results for the development of innovative snacks with improved functional properties.

Purple corn cobs (PCC) are normally discarded although containing valuable components of potential application in the development of foods and ingredients. Optimum extraction conditions for PCC bioactive compounds were defined considering maximum antioxidant activity (AA), anthocyanins concentration (TAC), and total phenolic content (TPC). The optimized PCC extract (Opt-PCC) was added to a maize-based extruded formulation, and the obtained products were compared with a control sample without extract regarding their color, TPC, AA, physical properties, water adsorption isotherms, thermal transitions, and microstructure. Besides significantly modifying the color of extrudates, the addition of Opt-PCC increased their physical stability since higher glass transition temperatures were obtained when compared to the control at similar water contents. Differences in water sorption behavior could be attributed to the hygroscopicity of the compounds present in the extract. The incorporation of Opt-PCC increased TPC and AA of the extrudates by 14 and 4 times, respectively. The results confirmed a significant increase in the functional properties and physical stability of the extrudates with the addition of Opt-PCC, encouraging its use as a natural ingredient in the development of novel foods with improved characteristics.

Solids-water interactions of corn and quinoa flours were evaluated through H-1 NMR, DSC, and water sorption isotherms. Glass transition temperature (T-g), observed by DSC, was better distinguished through FID signals, and correlated to water content through the Gordon and Taylor model. Enthalpy relaxations, identified by thermal analysis at 50-70 degrees C were studied through transverse relaxation times (T-2) measured after Hahn spin-echo sequence, which revealed a rearrangement of the biopolymers structures that cause immobilization of polymer chains and reduced mobility of water molecules with weak interactions with solids (lower T-22). The higher lipid content of quinoa flour was manifested after the CPMG sequence (T-2 approximate to 100 ms) and caused reduced hygroscopicity and T-g values compared with corn flour systems. H-1 NMR resulted efficient for assigning proton populations and understanding the changes in their distribution with temperature, analyzing glass transition and interpreting the implications of enthalpy relaxations processes in corn and quinoa flours.

The complex dependence of non-enzymatic browning development on processing conditions was analyzed in the production of maize snacks. The influence of the amount of water added to maize flour, toasting time, and toasting temperature on final water contents, HMF formation and CIELAB color parameters was evaluated by Response Surface Methodology. While L* values decreased continuously with increasing toasting temperature, the variables a* and b* showed maximum values at intermediate studied ranges of temperature and water contents, which is related to the complex interactions of the variables water content and temperature. The formation of HMF, as a marker of non-enzymatic browning reactions, was favored by low water contents and its concentration correlated with lower L* and b* but higher a* values. The optimum levels of toasting time, toasting temperature, and water addition for minimizing HMF concentration at which the snacks presented adequate color characteristics were determined. Finally, correlations between L*, b* and HMF were mathematically established to predict heat damage using these fast and non-destructive indicators to assure adequate processing and storage conditions.

The aim of this work was to evaluate the influence of partial replacement of maize flour by sub-valuated whole grain flours on the interactions and spatial distribution of the main components of extruded snacks. Non-destructive, fast spectroscopic and microscopic complementary tools were employed. All blends (composed by maize + 25% of pearl millet, red sorghum, quinoa or hairless canary seed) presented lower carbohydrate and lipid contents and higher protein, ash and dietary fiber levels than the control maize extrudates. These compositional changes were reflected in FT-MIR and FT-Raman spectra which also allowed detecting modifications at molecular level, such as protein agglomeration, amylose-lipids complexes formation and surface exposure of carotenoids. Tridimensional distribution and spatial arrangements of the main components in the extruded samples were studied through CLSM and XPS. These properties are related to quality aspects such as lipid oxidation sensitivity and textural properties. The combined FT-MIR, FT-Raman, CLSM and XPS were helpful tools to understand thermo-mechanical modifications of starch, proteins and lipids as a consequence of the extrusion process.

The increasing trend for using natural ingredients for nutraceuticals and phytopharmaceuticals development triggers the study of non-traditional sources for phytopharmaceuticals development, such as underexploited plants and agroindustrial wastes. The extraction, characterization and stabilization of bioactive compounds are intricate due to their low concentrations and their complex interactions in the vegetable matrix. New simple, ecological and efficient technologies are being developed to overcome the disadvantages of traditional extraction procedures, and many strategies should be developed to preserve their bioactivity. Oxidative reactions and protein glycation are two of the main deteriorative reactions affecting biological molecules and functionality loss in vitro and in vivo. Thus, efforts have been extended in search of edible plants with antioxidant or antiglycant properties. This chapter is an outcome of the CYTED Iberoamerican network 415RT0495, which task was to promote the valorization of subvaluated sources of bioactive compounds for food and medical uses.

Corn flour blends including 25 % of pearl millet, red sorghum, quinoa or canary seed flours, not deeply studied in the extrusion process of corn-based products, were used for extrudates production. Several physical, mechanical and microstructural properties, as well as water adsorption behavior were analyzed and compared to 100 % corn flour systems. The use of these flours increased the protein and fiber contents and modified several characteristics of the extrudates. The addition of quinoa and canary seed flour enhanced the expansion ratio and water absorption indexes, which could be attributed to their high protein and fiber contents. All extruded blends were darker than the control, with lower L* and higher a* values. A relationship between the microstructural characteristics, crystallinity and several physical properties was found, related to blends composition. The addition of flours with high crude fiber levels reduced hardness values and increased crystallinity of the products. The application of novel ingredients for human consumption is promissory for their re-valorization and further resource exploitation.

Chia (Salvia hispanica L.) and sesame (Sesamum indicum L.) oils are valorized for their health benefits and both are extensively used as ingredients in different food formulations and/or processes. Their retail prices are higher than those of other edible oils and might promote fraudulent adulterations. Spectroscopic methods associated to untargeted analysis are appropriate and faster than traditional techniques, requiring less time to prepare and run the samples. In the present study Fourier transform infrared spectroscopy was used in combination with one class partial least squares and soft independent modelling by class analogy to detect the presence of four possible adulterants: corn, peanut, soybean and sunflower oils, in four different proportions (pure + adulterant: 90 + 10, 95 + 5, 98 + 2 and 99 + 1, in volume). Untargeted approaches were successful in the detection of adulterated chia and sesame oils with acceptable prediction errors ranging between 1% and 5%.

This work describes a TLC-coupled bioautographic assay suitable for the separation and detection of apple polyphenol oxidase (PPO) inhibitors from natural extracts. PPO was immobilised in agar containing L-DOPA as substrate and 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) to enhance colour development. The inhibition was detected as white spots on reddish background. Minimum amount of PPO inhibitors detected was 0.0125 mu g of 4-hexylresorcinol, 0.025 mu g of ascorbic acid, 0.5 mu g of cysteine and 1 mu g of kojic acid. The assay was compatible with normal and reverse phase TLC systems and allows detecting compounds that directly had action on the enzyme as well as agents that could convert quinones back to their reduced form. The chromatographic run evidenced the different nature of enzymatic browning inhibitory compounds from garlic and onion extracts. Using natural enzymes will provide a fast and cheap alternative for target specific exploration of natural enzymatic inhibitors.

Spectral analysis employing multivariate techniques was employed to differentiate plain maize flours from formulations containing maize with added milled chia or quinoa seeds for producing cereal breakfast extrudates. The physicochemical changes of the enriched formulations due to processing stages and formulation were evaluated by using FTIR and chemometric analysis, which allowed a rapid and non-destructive discrimination between sample processing and compositional aspects. Specific IR frequencies were selected which provided highest sample discrimination. Selected IR absorbance relationships at those specific wavenumbers were useful to track changes promoted by extrusion for carbohydrates, proteins, and lipids. The complexes between amylose and lipids, that takes place during extrusion, underwent distinctive changes as confirmed by XRD. The crystallinity loss, after extrusion (with an average value of 50%), shows evidence of amylose-lipid complexes formation of type Eh and Vh. Correlations between the textural behavior, composition, and selected FTIR indices were obtained.

During toasting, the last stage of corn flake production, Maillard reaction takes place, favored by the high temperature and low water content. The cooking formulation ingredients influence color and flavor of the final product and, therefore, consumer acceptance. However, some undesirable components are also formed. The impact of cooking formulation and toasting time on color development and on the formation of chemical markers was investigated. Samples (flakes) were equilibrated at water activity ( a w ) 0.8 and toasted at 230°C. After extraction of fluorescent pigments with pronase, fluorescence, absorbance at 420 nm, and furfurals analysis was performed. Sucrose showed a synergistic interaction with malt and salt. Formulation highly affected the amount of 5‐hydroxymethylfurfural and furfural formed. L* and a* were sensitive variables to measure overall browning reaction. These results allow for further understanding of the influence of formulation used during cooking and would help to mitigate the formation of undesirable compounds.

The objective of this study was to evaluate the impact of process and formulation on individual carotenoid loss in traditionally prepared cornflakes and those prepared by extrusion. The first step in the traditional process (maize grits cooking) promoted a 60% lutein content reduction and 40% in zeaxanthin loss, showing lutein more susceptibility to isomerization and decomposition. After toasting, the last step, the total loss averaged 80% for both compounds. The extruded maize in a plain formulation showed a 35% lutein and zeaxanthin reduction. However, in samples containing quinoa the decrease reached 60%, and the major loss (80%) was found in chia-containing formulations. Correlations between the color coordinate b∗, total and individual carotenoid content, were obtained. It is of a major importance that the efforts to increase carotenoid content in raw materials are complemented with attempts to reduce the losses during processing.