Analysis of Volatile Hydrocarbons (Pentene Dimers and Terpenes) in Extra Virgin Olive Oil: Optimization by Response Surface Methodology and Validation of HS-SPME-GC-MS Method.

A head space-solid phase microextraction-gas chromatography-mass spectrometery (HS-SPME-GC-MS) method for the simultaneous analysis of pentene dimers from lipoxygenase (LOX) pathway, monoterpenes, and sesquiterpenes in extra virgin olive oil (EVOO) was proposed. A Doehlert design was performed; the conditions of the HS-SPME preconcentration step (extraction temperature, extraction time, sample amount, and desorption time) were optimized by response surface methodology, allowing defining the method operable design region. A quantitative method was set up using the multiple internal standard normalization approach: four internal standards were used, and the most suitable one was selected for area normalization of each external standard. The quantitative method was successfully validated and applied to a series of monocultivar EVOOs. This is the first paper in which a quantitative method using commercial standards has been proposed for the analysis of an important class of molecules of EVOO such as pentene dimers. The optimized method is suitable for routine analysis aimed at characterizing high quality EVOOs.

Proso Millet (Panicum miliaceum L.) as Alternative Source of Starch and Phenolic Compounds: A Study on Twenty-Five Worldwide Accessions.

Proso millet has been proposed as an effective anti-diabetic food thanks to the combined action of polyphenols and starch. This study aimed to characterize the chemical composition of twenty-five accessions, in order to enhance this cereal as an alternative to available starch for food applications or to propose new food ingredients with health benefits. Proso millet contained a high percentage of starch, reaching values of 58.51%. The amylose content showed high variability, with values ranging from 1.36 to 42.70%, and significantly higher values were recorded for the white accessions than for the colored ones. High-resistant starch content (13.41-26.07%) was also found. The HPLC-MS analysis showed the same phenolic pattern in all the samples. Cinnamic acids are the most abundant compounds and significant differences in their total content were found (0.69 to 1.35 mg/g DW), while flavonoids were only detected in trace amounts. Statistical results showed significantly higher antiradical activity in the colored accessions than in the white ones.

Millet Fermented by Different Combinations of Yeasts and Lactobacilli: Effects on Phenolic Composition, Starch, Mineral Content and Prebiotic Activity.

Millet is the sixth-highest yielding grain in the world and a staple crop for millions of people. Fermentation was applied in this study to improve the nutritional properties of pearl millet. Three microorganism combinations were tested: Saccharomyces boulardii (FPM1), Saccharomyces cerevisiae plus Campanilactobacillus paralimentarius (FPM2) and Hanseniaspora uvarum plus Fructilactobacillus sanfranciscensis (FPM3). All the fermentation processes led to an increase in minerals. An increase was observed for calcium: 254 ppm in FPM1, 282 ppm in FPM2 and 156 ppm in the unfermented sample. Iron increased in FPM2 and FPM3 (approx. 100 ppm) with respect the unfermented sample (71 ppm). FPM2 and FPM3 resulted in richer total phenols (up to 2.74 mg/g) compared to the unfermented sample (2.24 mg/g). Depending on the microorganisms, it was possible to obtain different oligopeptides with a mass cut off ≤10 kDalton that was not detected in the unfermented sample. FPM2 showed the highest resistant starch content (9.83 g/100 g) and a prebiotic activity on Bifidobacterium breve B632, showing a significant growth at 48 h and 72 h compared to glucose (p < 0.05). Millet fermented with Saccharomyces cerevisiae plus Campanilactobacillus paralimentarius can be proposed as a new food with improved nutritional properties to increase the quality of the diet of people who already use millet as a staple food.

Co-milling of sound olives with fresh chili peppers improves the volatile compound, capsaicinoid and sensory profiles of flavoured olive oil with respect to the typical infusion.

In the context of the olive oil flavoured with chili peppers, the aim of this study was to compare co-milling of sound olives and fresh chili peppers at mill scale to infusion of dried chili peppers in oil, using the same batch of olives for all oils. Capsaicinoids by HPLC-DAD, volatile profile by HS-SPME-GC-MS and HS-GC-IMS and sensory profile were characterized. Capsaicinoids were statistically higher in oils prepared with green (52.0-68.0 mg/kg) than red (48.0-60.2 mg/kg) chili peppers. Oils flavoured by infusion showed higher contents of volatile compounds linked to defects such as acetic acid, with winey/vinegary sensory defect (median, 1.72-2.02) and no fresh pepper flavour. Oils prepared by co-milling resulted rich in the typical esters of chili pepper (6.175 and 4.156 mg/kg with green and red chili peppers, respectively), with pleasant hotness sensation and fresh pepper flavour. Overall, the co-milling approach allowed obtaining flavoured samples with improved sensory quality.

Industrial drying for agrifood by-products re-use: Cases studies on pomegranate peel (Punica granatum L.) and stoned olive pomace (pâtè, Olea europaea L.).

The effect of industrial drying processes on phenols and polysaccharides of olive pomace (pâté) and pomegranate peel was studied, with the aim to re-use pomegranate and olive oil by-products. Pomegranate peel (Wonderful and G1 varieties) was oven-dried at different temperatures, taking into account peel thickness and size. Pâté was freeze-dried and oven dried at 50-110 °C, at lab scale; then, an industrial drying system (150 °C) was compared to freeze-drying. All dried samples were analyzed in terms of phenolic and polysaccharides compounds. Drying at room temperature of small pieces of pomegranate peel guaranteed the highest humidity removal and recovery of phenols. Sugar analysis, DLS and 1H NMR confirmed that polysaccharide fractions were not significantly affected by the highest drying temperatures (42 °C for pomegranate, 150 °C for pâté). The two drying procedures at large scale were suitable for avoiding degradation of phenols, maintaining the same profiles of the corresponding freeze-dried samples.

Pomegranate peel as a promising source of pectic polysaccharides: A multi-methodological analytical investigation.

Polysaccharides from pomegranate peel (Wonderful and Purple Queen® varieties) were extracted by hot water and fractionated using ethanol. Three fractions (F1-F2-F3) were obtained for each sample. Polysaccharides' yield was higher for Purple Queen®: 13% dw. Polysaccharides of the three fractions were characterized by size exclusion chromatography (SEC), dynamic light scattering (DLS), 1H NMR, methylation, and acylation degree. Differently from SEC, DLS highlighted some differences between the polysaccharides's molecular sizes of the fractions. The highest methylation and acylation degree was observed for F3 of Purple Queen®: 74.0% and 18.6%, respectively. The percentage of galacturonic acid confirmed the presence of pectin in almost all the fractions recognized as homogalacturonan. Arabinan and arabinogalactan were also found in all the collected F3 samples, although in different proportions. The stepwise fractionation process followed by a multi-methodological analytical investigation was helpful to improve the knowledge of the pectic polysaccharides of pomegranate.

Phenolic compounds and polysaccharides in the date fruit (Phoenix dactylifera L.): Comparative study on five widely consumed Arabian varieties.

The study analysed polysaccharides and phenolic compounds in widely consumed but little studied date fruits varieties such as Sukkari, Ajwa, Segae, Barrny and Khalas harvested at Tamr stage. The total phenols were in similar amount in the five varieties and ranged from 20 to 50 mg/100 g DW. The decoction and successive centrifugation made it possible to collect two main polysaccharide fractions for all the selected fruits. For each variety the first fraction was more abundant, with a lower swelling capacity and a higher amount of galacturonic acid (28.3% to 40.1%). The second fraction was only soluble in alkaline solution, with an average galacturonic acid content of only 17%. The different structure of the two polysaccharide fractions was also confirmed by the composition in neutral sugars and the degrees of methylation and acetylation. The proposed extraction procedure could be applied for larger scale extraction of the date fruit polysaccharides.

Steryl ferulates composition in twenty-two millet samples: Do "microwave popping" and fermentation affect their content?

Climate change has led to rediscovery of minor drought tolerant grains such as millet. Among its bioactive molecules, steryl ferulates have been poorly explored. Steryl ferulates composition of was investigated by high performance liquid chromatography-diode array detector-tandem mass spectrometry and high resolution tandem mass spectrometry in twenty-two millet samples and also in some fermented and microwave heated samples. Six compounds were found in Panicum, Pennisetum, Eleusine and Setaria genera, with a prevalence of campestanyl and sitostanyl ferulate. The lowest steryl ferulates content was found in Panicum, with values ranging from 2.98 ± 0.04 µg/g to 8.72 ± 0.41 µg/g. Foxtail millet and finger millet showed the highest amount with 46.07 ± 5.20 µg/g and 85.29 ± 4.30 µg/g, respectively. As for pearl millet, microwave heating and fermentation increased steryl ferulates by two (33.77 ± 0.88 µg/g) and five (75.83 ± 1.25 µg/g) times, with respect to the untreated sample. Microwave heating and fermentation could be used to increase steryl ferulates in millet.

1H NMR and HPLC-DAD-MS for the characterization of ellagitannins and triterpenoids of less investigated Anogeissus leiocarpus DC (Combretaceae) stem bark.

Anogeissus leiocarpus DC is an evergreen tree, widely distributed in Asia and Africa. The stem bark is used in traditional medicine, and as chewing sticks and infusion. Nowadays, it is becoming increasingly important to define the phytochemical profile of less studied edible plants. Aim of this research was a first complete characterization of ellagitannins and triterpenoids profiles by HPLC-DAD-MS and 1H NMR and analyses. A total of 59 compounds were identified including 43 ellagitannins and 16 triterpenoids, mainly oleane derivatives and glycosylated forms. Among ellagitannins, roburin, vescalin and castalin were found for the first time. Tannins accounted for 38.9% whereas triterpenoids were 4.8%, both estimated on dry decoction. The decoction was preliminary tested against osteoarthritis in rats. The characterization of the main phytochemicals of Anogeissus leiocarpus DC stem bark decoction is a necessary step to evaluate nutraceutical properties, paving the way for possible food applications of this plant.

Phenolic Compounds and Triterpenes in Different Olive Tissues and Olive Oil By-Products, and Cytotoxicity on Human Colorectal Cancer Cells: The Case of Frantoio, Moraiolo and Leccino Cultivars (Olea europaea L.).

Phenolic and triterpenoid compounds of the olive tree are recognized as having a key role in health promotion, thanks to their multiple protective actions in humans. To expand the source of these bioactive compounds, the phenolic and triterpenoid profiles of leaf, branch, destoned fruit, destoned pomace, shell, seed, and extra virgin olive oil from the Frantoio, Leccino, and Moraiolo olive cultivars were simultaneously characterized by HPLC-DAD-MS. Overall, 43 molecules were quantitated and expressed on the obtained dry extracts. Oleuropein was mainly concentrated in branches (82.72 g/kg), fruits (55.79 g/kg), leaves (36.71 g/kg), and shells (1.26 g/kg), verbascoside (4.88 g/kg) in pomace, and nüzhenide 11-methyl oleoside (90.91 g/kg) in seeds. Among triterpenoids, which were absent in shells, the highest amount of oleanolic acid was found in olive leaves (11.88 g/kg). HCT-116 colorectal cells were chosen to assess the cytotoxicity of the dry extract, using the phytocomplex from Frantoio, which was the richest in phenols and triterpenoids. The IC50 was also determined for 13 pure molecules (phenols and terpenoids) detected in the extracts. The greatest inhibition on the cell's proliferation was induced by the branch dry extract (IC50 88.25 µg/mL) and by ursolic acid (IC50 24 µM). A dose-dependent relationship was observed for the tested extracts.

Food by-products valorisation: Grape pomace and olive pomace (pâté) as sources of phenolic compounds and fiber for enrichment of tagliatelle pasta.

Wine and olive oil making by-products are rich sources of bioactive compounds suitable for new healthy recipes of staple foods. In this study, the profile of pasta (tagliatelle) fortified with 7% of grape pomace (GP) or olive pomace (pâté, OP) was studied, focusing on phenolic compounds after cooking. The enriched tagliatelle retained the same monoglycosylated and acetylated anthocyanins found in grape pomace. The fortified tagliatelle with a new milling by-product called pâté retained hydroxytyrosol after cooking (6.6 mg/100 g). In both the two types of enriched tagliatelle the fiber content increased of approx. 3%, while the added phenols retained after cooking by tagliatelle fortified with GP and OP were 6.21 mg/100 g and 9 mg/100 g, respectively. The fortified tagliatelle retained a good cooking resistance and a good texture after cooking, thus enhancing the nutritional profile of pasta, a staple food usually characterized by a negligible amount of phenolic compounds and fiber.

A Study on the Biodiversity of Pigmented Andean Potatoes: Nutritional Profile and Phenolic Composition.

The characterization of six varieties of native Andean potatoes with a wide biodiversity in tuber shape, flesh, and skin color was performed, through the determination of their proximate composition, mineral content, and phenolic profile. Minerals concentration revealed significant genotypic variation. Potassium was the most abundant element in all varieties, ranging from 7272.9 to 13,059.9 µg/g and from 12,418 to 17,388.6 µg/g dried weight for the flesh and skin samples, respectively. Iron content was relevant, ranging from 20.5 to 39.9 µg/g and from 112.2 to 288.8 µg/g dried weight in flesh and skin samples, respectively. Phenolic compounds were consistently higher in the skin than in the flesh. The total content varied greatly from 19.5 to 2015.3 µg/g and from 1592.3 to 14807.3 µg/g dried weight for flesh and skin tissues, respectively. 5-caffeoylquinic acid was 74% of the total phenolic acids. Different pattern of anthocyanins was found, depending on the color of the variety; the red genotypes contained predominantly pelargonidin derivatives, while the purple samples had petunidin as a major anthocyanidin. This study increases the knowledge of the composition of the local Andean varieties (which are only scarcely studied so far), helping to enhance these genotypes and the conservation of biodiversity.

Does Fermentation Really Increase the Phenolic Content in Cereals? A Study on Millet.

Millet is underutilized in Europe, despite its advantages compared to other common cereals. In Asia and Africa, millet is mainly eaten in fermented form; its consumption has beneficial properties on human health. Three millet batches were compared in terms of free and bound phenols by High Performance Liquid Chromatography-Diode Array Detector-Mass Spectrometry (HPLC-DAD-MS). The richest one in terms of bound phenols was selected for testing via a basic (0.1 M NaOH) and an acidic (1.2 M H2SO4) hydrolysis, in which 149.3 and 193.6 mg/100 g of phenols were recovered, respectively. The ability of fermentation, with yeast and Lactobacilli, to increase the content of phenolic compounds was verified. Five withdrawalswere performed to verify the influence of fermentation time on the total phenolic content. The greatest phenolic content was observed after 72 h. Fermentation increased the cinnamic acids and flavonoids contents by approximately 30%. Vitexin and vitexin 2″-O-rhamnoside contents were significantly higher in the fermented millet; these compounds partially inhibit the protein tyrosine phosphatase enzyme, which is overexpressed in type-2 diabetes. A molecular dynamic simulation showed the two flavonoids to be allosteric inhibitors. The phenolic extract from fermented millet demonstrated a higher level of antioxidant protection on human erythrocytes by ex vivo cellular antioxidant activity in red blood cells. In this context, functional foods based on fermented millet could represent a new trend in European markets.

Characterization of Arils Juice and Peel Decoction of Fifteen Varieties of Punica granatum L.: A Focus on Anthocyanins, Ellagitannins and Polysaccharides.

Pomegranate is receiving renewed commercial and scientific interest, therefore a deeper knowledge of the chemical composition of the fruits of less studied varieties is required. In this work, juices from arils and decoctions from mesocarp plus exocarp were prepared from fifteen varieties. Samples were submitted to High Performance Liquid Chromatography-Diode Array Detector-Mass Spectrometry, spectrophotometric and colorimetric CIEL*a*b* analyses. Antioxidant, antiradical and metal chelating properties, inhibitory activity against tyrosinase and α-amylase enzymes were also evaluated. All varieties presented the same main phenols; anthocyanins and ellagitannins were widely variable among varieties, with the richest anthocyanin content in the juices from the Wonderful and Soft Seed Maule varieties (approx. 660 mg/L) and the highest ellagitannin content in the peel of the Black variety (approx. 133 mg/g dry matter). A good correlation was shown between the colour hue and the delphinidin/cyanidin ratio in juices (R2 = 0.885). Total polysaccharide yield ranged from 3% to 12% of the peels' dry weight, with the highest content in the Black variety. Decoctions (24.44-118.50 mg KAE/g) showed better in vitro antioxidant properties and higher inhibitory capacity against tyrosinase than juices (not active-16.56 mg KAE/g); the inhibitory capacity against α-amylase was similar and quite potent for juices and decoctions. Knowledge about the chemical composition of different pomegranate varieties will allow for a more aware use of the different parts of the fruit.

Optimized hydrolytic methods by response surface methodology to accurately estimate the phenols in cereal by HPLC-DAD: The case of millet.

Extraction of free and bound phenols from millet in acidic and basic hydrolytic conditions were compared for the first time. Acidic hydrolysis was able to extract the highest amount of total phenolic compounds (up to 178 mg/100 g) while the basic hydrolysis underestimates the phenolic concentration. Our findings pointed out for the first time that methyl ferulate is naturally present as bound phenol in millet. Response Surface Methodology was then applied to both acidic and basic hydrolytic extractive conditions: the acidic procedure, optimized in terms of extractive time and temperature and concentration of the acidic mean, gave the best results, allowing definition of Method Operable Design Region and quantitation of the total amount of phenols in millet samples in a single extractive step. This optimized method is suitable for further accurate investigations of the typical phenols of the numerous varieties of this recently re-discovered minor cereal.

Study on a Fermented Whole Wheat: Phenolic Content, Activity on PTP1B Enzyme and In Vitro Prebiotic Properties.

Fermented cereals, staple foods in Asia and Africa, are recently receiving a growing interest in Western countries. The object of this work is the characterization of a fermented wheat used as a food ingredient and dietary supplement. To this aim, the phenolic composition, the activity on protein tyrosine phosphatase 1B (PTP1B), an enzyme overexpressed in type-II diabetes, the in vitro prebiotic properties on Lactobacillus reuteri and the microbial composition were investigated. Basic and acidic hydrolysis were tested for an exhaustive recovery of bound phenols: the acidic hydrolysis gave best yields. Methyl ferulate and neocarlinoside were identified for the first time in wheat. The inhibitory power of the extracts of several batches were investigated on PTP1B enzyme. The product was not able to inhibit the enzyme, otherwise, for the first time, a complete inhibition was observed for schaftoside, a major C-flavonoid of wheat. The microbial composition was assessed identifying Lactobacillus, Enterococcus, and Pediococcus as the main bacterial species. The fermented wheat was a suitable substrate for the grown of L. reuteri, recognized for its health properties in the human gut. The proposed method for phenols is easier compared to those based on strong basic hydrolysis; our results assessed the bound phenols as the major fraction, differently from that suggested by the literature for fermented cereals.