Estudio de perfilación de metabolitos secundarios de extractos vegetales obtenidos mediante técnicas y estrategias no convencionales para el desarrollo de alimentos funcionales explotando residuos alimentarios

En esta tesis doctoral se desarrolló en el entorno de la economía verde y el reciclaje de residuos alimentarios estudiando diferentes técnicas de extracción no convencionales. En primer lugar, se aislaron polifenoles de matrices complejas producto del desperdicio de alimentos, como son las hojas de Moringa oleifera, mediante extracción asistida por ultrasonidos (UAE). El enfoque de diseño experimental permitió optimizar las mejores condiciones de extracción (MeOH:H2O 50:50, v/v, proporcion 1:60 Sólido/Líquido (S/L), 60°C, 60 min) para obtener un contenido fenólico total (TPC) de 13,4 mg expresados como equivalentes de ácido gálico (GAE)/g de materia seca. Por lo tanto, la caracterización y cuantificación de la fracción de flavonol se llevó a cabo mediante un UHPLC-MS/MS (Cromatografía líquida de ultra alta resolución-espectrometría de masas en tándem) y HPLC-DAD (Cromatografía líquida de alta resolución-espectrofotometría de fila de diodos). Los flavonoles más abundantes fueron las formas glicídicas de quercetina y kaempferol (que van desde 216,4 μg/g de MS de quercetina 3-O-ramnosido a 293,9 μg/g de materia seca de quercetina 3-O- (6 ″ -O-malonil) -β- D-glucósido). Además, las hojas de Lycium barbarum se estudiaron comparando los UAE con la extracción asistida por microondas (MAE), utilizando solventes alcohólicos e hidroalcohólicos. Los UAE metanólicos resultaron en la extracción más eficiente, confirmando que tanto el solvente como la técnica empleada influyeron en la extracción de compuestos bioactivos. Por lo tanto, las hojas de L. barbarum y L. chinense cultivadas en el centro de Italia se extrajeron con la técnica UAE con metanol. La actividad antioxidante se investigó mediante ensayos de captación de radicales libres 10 in vitro como DPPH, ABTS y FRAP. Los análisis cromatográficos (HPLC-DAD) demostraron que el ácido clorogénico y la rutina son los compuestos fenólicos más importantes en L. barbarum, y el ácido clorogénico y el tirosol en L. chinense. Finalmente, se realizó el análisis estadístico multivariante para diferenciar las muestras procedentes de Lycium spp. Estos estudios pusieron de manifiesto la importancia en la elección de la técnica de extracción para aislar compuestos fenólicos y también confirmaron a M. oleifera, L. barbarum y L. chinense como matrices vegetales ricas en antioxidantes, normalmente descartadas por la industria agrícola-alimentaria. Por tanto, estos resultados mejoraron el conocimiento actual sobre la recuperación de compuestos bioactivos a partir de residuos alimentarios para desarrollar productos de valor añadido como nutracéuticos y alimentos funcionales. Por esta razón, se investigó el efecto del MAE doméstico sobre el contenido fenólico y la actividad antioxidante de los extractos de hojas de L. barbarum para estudiar un alimento funcional como las bebidas a base de hierbas. Se realizó un enfoque de diseño experimental considerando la relación S/L, el tiempo de irradiación y la potencia de microondas como variables independientes sobre las respuestas del TPC, la actividad antioxidante y el contenido de ácido clorogénico. Los resultados demostraron que las respuestas se vieron influidas positivamente por la relación S/L y el tiempo, mientras que la potencia de microondas se correlacionó inversamente con las respuestas investigadas. Esta investigación reveló que las condiciones de extracción por microondas 11 deben monitorearse cuidadosamente para obtener extractos acuosos ricos en compuestos bioactivos con alta actividad antioxidante. La comparación con los métodos tradicionales domésticos (decocción e infusión) mostró un contenido fenólico y una actividad antioxidante inesperados al resultar más bajos para el extracto realizado con MAE. De hecho, se encontró que la infusión de hojas de L. barbarum tenía las mejores propiedades funcionales. Este estudio contribuyó a crear conciencia de que las condiciones de preparación de los extractos de hierbas en el hogar afectan fuertemente el estado fisológico y la salud de los consumidores. Hoy en día es de gran interés no solo la extracción de compuestos bioactivos a partir de residuos vegetales, sino también el estudio de su potencial inhibitorio frente a las enzimas digestivas. De hecho, se estudió la actividad inhibidora del extracto metanólico de UAE de hojas de L. barbarum sobre la α-amilasa del páncreas porcino. También se investigó la actividad inhibidora de los ácidos fenólicos aislados. Los ácidos fenólicos se identificaron mediante cromatografía líquida/espectrometría de masas de tiempo de vuelo cuadrupolo de masa precisa (LC-QTOF/MS). Los ácidos clorogénico y salicílico representaron los ácidos fenólicos más abundantes en el extracto de hoja de L. barbarum. El efecto inhibidor frente a la α-amilasa se determinó para compuestos individuales (los ácidos clorogénico, salicílico y cafeico dieron la mayor inhibición) mediante un ensayo in vitro. El extracto de hoja de L. barbarum mostró un efecto inhibidor sobre la α-amilasa apreciable de una manera dependiente de la concentración. Además, los estudios de acoplamiento de los ácidos fenólicos considerados en el sitio activo de la α-amilasa sugirieron un modo de unión conservado que se estabiliza principalmente a través de enlaces H e interacciones de apilamiento π-π. En cuanto al desperdicio de frutas, el orujo de manzana es uno de los desperdicios alimentarios más abundantes que se producen a nivel mundial. Consiste en la parte sólida de la pulpa, piel, pepitas y pecíolo que se obtiene durante la elaboración del zumo de manzana o de la sidra. Así, se produjo orujo de manzana Red Delicious a escala de laboratorio y se realizaron diferentes técnicas de extracción no convencionales para aislar compuestos fenólicos, como UAE, Extracción Ultraturrax (UTE), Extracción Acelerada por Solvente (ASE) y Campo Eléctrico Pulsado (PEF). La UAE demostró ser la técnica de extracción de polifenoles más eficiente cuando se llevó a cabo con EtOH:H2O (50:50, v/v). La floridzina, el principal compuesto fenólico en la manzana, se analizó mediante análisis cromatográfico LC-QTOF/MS, y la extracción con ASE y EtOH:H2O (30:70, v/v) a 40°C, demostró ser la técnica más eficaz en la obtención de floridzina. Los resultados obtenidos confirman que la floridzina podría considerarse un biomarcador para determinar la calidad de numerosos productos de manzana. Por tanto, el orujo de manzana podría considerarse otro desecho de fruta interesante para desarrollar un producto de valor añadido como son alimentos funcionales y nutracéuticos

Invited review: Authentication of milk by direct and indirect analysis of triacylglycerol molecular species.

Milk, milk-based products, and milk derivatives represent an important group of food commodities, with high nutritional value and widely consumed by large segment of consumers, including pregnant women, newborns, children, and the elderly. Food authentication is a rapidly growing field because of increasing consumer awareness regarding food quality and safety. This review attempts to critically summarize the status of direct and indirect analysis of the molecular species of triacylglycerols (TAG) used to assess the authenticity of milk. Identification and quantification of TAG molecular species in milk fat can be accurately performed even though analytical approaches focused on fraud evaluation should be developed. Recent analytical breakthroughs and novel techniques are discussed, along with their applications in milk authentication

Phenol Profiling and Nutraceutical Potential of Lycium spp. Leaf Extracts Obtained with Ultrasound and Microwave Assisted Techniques

In recent years, agricultural and industrial residues have attracted a lot of interest in the recovery of phytochemicals used in the food, pharmaceutical, and cosmetic industries. In this paper, a study on the recovery of phenol compounds from Lycium spp. leaves is presented. Ultrasound‐assisted extraction (UAE) and microwave‐assisted extraction (MAE) have been used with alcoholic and hydroalcoholic solvents. Methanolic UAE was the most successful technique for extracting phenols from Lycium leaves, and we used on leaves from L. barbarum and L. chinense cultivated in Italy. The extracts were then characterized as regards to the antioxidant properties by in vitro assays and the phenol profiling by a high performance liquid chromatography‐diode array detector (HPLC‐DAD). Chlorogenic acid and rutin were the main phenol compounds, but considerable differences have been observed between the samples of the two Lycium species. For example, cryptochlorogenic acid was found only in L. barbarum samples, while quercetin‐3‐O‐rutinoside‐7‐O‐glucoside and quercetin‐3‐O‐sophoroside‐7‐O‐rhamnoside only in L. chinense leaves. Finally, multivariate statistical analysis techniques applied to the phenol content allowed us to differentiate samples from different Lycium spp. The results of this study confirm that the extraction is a crucial step in the analytical procedure and show that Lycium leaves represent an interesting source of antioxidant compounds, with potential use in the nutraceutical field

Phenolic Acids from Lycium barbarum Leaves: In Vitro and In Silico Studies of the Inhibitory Activity against Porcine Pancreatic α-Amylase

Nowadays, bioactive compounds from vegetable food and waste are of great interest for their inhibitory potential against digestive enzymes. In the present study, the inhibitory activity of methanolic extract from Lycium barbarum leaves on porcine pancreas α-amylase has been studied. The α-amylase inhibitory activity of the constituent phenolic acids was also investigated. The leaves were extracted by ultrasound-assisted method, one of the most efficient techniques for bioactive extraction from plant materials, and then the phenolic acids were identified by Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) Liquid Chromatography/Mass Spectrometry (LC/MS). Chlorogenic and salicylic acids were the most abundant phenolic acids in L. barbarum leaf extract. The inhibitory effect against α-amylase, determined for individual compounds by in vitro assay, was higher for chlorogenic, salicylic, and caffeic acids. L. barbarum leaf extract showed an appreciable α-amylase inhibitory effect in a concentration-dependent manner. Docking studies of the considered phenolic acids into the active site of α-amylase suggested a conserved binding mode that is mainly stabilized through H-bonds and π-π stacking interactions

Varietal Authentication of Extra Virgin Olive Oils by Triacylglycerols and Volatiles Analysis

In recent years, there is an increasing interest in high-quality extra virgin olive oils (EVOOs) produced from local cultivars. They have particular chemical/organoleptic characteristics and are frequently subjected to fraud, whereby the control of quality requires a powerful varietal check. In the present research, triacylglycerols (TAGs) and volatiles have been studied as chemical markers for the authentication of EVOO samples from four Italian varieties of Olea europea (Dolce Agogia, Frantoio, Leccino, and Moraiolo). The monocultivar EVOO samples have been subjected to a chemical–enzymatic chromatographic method in order to perform a stereospecific analysis, an important procedure for the characterization of TAG of food products. The results, combined with chemometric analysis (linear discriminant analysis, LDA), were elaborated in order to classify Italian EVOO monocultivar samples. In accordance with the total and intrapositional fatty acid (FA) composition of TAG fraction, the results were allowed to carry out a varietal discrimination. In addition, volatile compounds were also determined by solid-phase micro-extraction gas chromatography-mass spectrometry analysis. All EVOO samples were correctly classified when TAG stereospecific data and volatile results were elaborated by the LDA procedure, even if volatile compounds showed a higher discriminant power

Extraction of Phenolic Compounds from Fresh Apple Pomace by Different Non-Conventional Techniques

Red Delicious apple pomace was produced at laboratory scale with a domestic blender and different non-conventional extraction techniques were performed to isolate phenolic compounds, such as ultrasound-assisted extraction (UAE), ultraturrax extraction (UTE), accelerated solvent extraction (ASE) and pulsed electric field (PEF) extraction pre-treatment. Total phenolic content (TPC) was determined by Folin–Ciocalteu assay. Phloridzin, the main phenolic compound in apples, was determined by chromatographic analysis Q-TOF-LC/MS. The results obtained with these techniques were compared in order to identify the most efficient method to recover polyphenols. The highest value of TPC (1062.92 ± 59.80 µg GAE/g fresh apple pomace) was obtained when UAE was performed with EtOH:H2O (50:50, v/v), while ASE with EtOH:H2O (30:70, v/v) at 40 °C and 50% of flush was the most efficient technique in the recovery of phloridzin. The concentration of the main phenolic compounds ranged from 385.84 to 650.56 µg/g fresh apple pomace. The obtained results confirm that apple pomace represents an interesti-ng by-product, due to the presence of phenolic compounds. In particular, phloridzin could be considered a biomarker to determine the quality of numerous apple products. Therefore, this research could be a good starting point to develop a value-added product such as a functional food or nutraceutical

Is the Household Microwave Recommended to Obtain Antioxidant-Rich Extracts from Lycium barbarum Leaves?

Nowadays, much interest is devoted to the extraction of plant materials, considering also their waste and by-products, to obtain antioxidant-rich products. The effect of household microwave-assisted extraction (MAE) on the phenolic content and antioxidant activity of Lycium barbarum leaf extracts was investigated. An experimental design approach was adopted considering solid/liquid ratio (1, 3, and 5 g of leaves in 150 mL water), irradiation time (1, 3, and 5 min), and microwave power (300, 400, and 500 W) as independent variables. These three factors and their interactions were studied to evaluate the effect of MAE conditions on the responses of total phenolic content, antioxidant activity, and chlorogenic acid content. The results showed that the analytical parameters were positively influenced by the solid/liquid ratio and time. On the contrary, microwave power was inversely correlated with the investigated responses. This research revealed that microwave extraction conditions should be carefully monitored to obtain bioactive-rich aqueous extracts with high antioxidant activity. A comparison with household traditional methods showed an unexpected lower phenolic content and antioxidant activity for MAE extract in respect to the decoction and infusion. In fact, it was found that L. barbarum leaf infusion had the best functional properties, regarding the investigated characteristics. The outcome of this study has implications for raising awareness that household preparation conditions strongly affect the health properties of herbal extracts

Phenol Profiling and Nutraceutical Potential of Lycium spp. Leaf Extracts Obtained with Ultrasound and Microwave Assisted Techniques

In recent years, agricultural and industrial residues have attracted a lot of interest in the recovery of phytochemicals used in the food, pharmaceutical, and cosmetic industries. In this paper, a study on the recovery of phenol compounds from Lycium spp. leaves is presented. Ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE) have been used with alcoholic and hydroalcoholic solvents. Methanolic UAE was the most successful technique for extracting phenols from Lycium leaves, and we used on leaves from L. barbarum and L. chinense cultivated in Italy. The extracts were then characterized as regards to the antioxidant properties by in vitro assays and the phenol profiling by a high performance liquid chromatography-diode array detector (HPLC-DAD). Chlorogenic acid and rutin were the main phenol compounds, but considerable differences have been observed between the samples of the two Lycium species. For example, cryptochlorogenic acid was found only in L. barbarum samples, while quercetin-3-O-rutinoside-7-O-glucoside and quercetin-3-O-sophoroside-7-O-rhamnoside only in L. chinense leaves. Finally, multivariate statistical analysis techniques applied to the phenol content allowed us to differentiate samples from different Lycium spp. The results of this study confirm that the extraction is a crucial step in the analytical procedure and show that Lycium leaves represent an interesting source of antioxidant compounds, with potential use in the nutraceutical field

Untargeted Metabolomics to Evaluate the Stability of Extra-Virgin Olive Oil with Added <i>Lycium barbarum</i> Carotenoids during Storage

A carotenoid-rich extract from Lycium barbarum L. was added to extra virgin olive oil (EVOO), obtaining a carotenoid-enriched oil (EVOOCar). The oxidative stability of EVOO and EVOOCar was evaluated during long-term storage of 28 weeks at room temperature, by measuring some classical parameters (acidity and peroxide values, spectrophotometric coefficients, fatty acid composition) and the content of minor compounds (i.e., &#945;-tocopherol and lutein). At the end of the storage, higher content (p &lt; 0.01) of &#945;-tocopherol in EVOOCar in respect to EVOO were observed. Zeaxanthin dipalmitate, the most abundant carotenoid compound of Goji berries, decreased slightly (p &lt; 0.05) in EVOOCar until the end of the storage. In regard to polyphenols, an ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC/QTOF-MS) using untargeted metabolomics was carried out. This latter approach discriminated the two oil samples during long-term storage, allowing to identify also the phenolic classes most exposed to significant variations during storage (i.e., mainly lignans and flavones). Besides, the addition of Goji carotenoids preserved the stability of tyrosol equivalents in EVOOCar during long-term storage. These results highlighted that the enrichment of EVOO with a carotenoid-rich extract can improve the shelf-life and nutritional value of added-oil, protecting EVOO natural antioxidants during long-term storage