Effects of Garlic on Breast Tumor Cells with a Triple Negative Phenotype: Peculiar Subtype-Dependent Down-Modulation of Akt Signaling.

Breast cancer includes tumor subgroups with morphological, molecular, and clinical differences. Intrinsic heterogeneity especially characterizes breast tumors with a triple negative phenotype, often leading to the failure of even the most advanced therapeutic strategies. To improve breast cancer treatment, the use of natural agents to integrate conventional therapies is the subject of ever-increasing attention. In this context, garlic (Allium sativum) shows anti-cancerous potential, interfering with the proliferation, motility, and malignant progression of both non-invasive and invasive breast tumor cells. As heterogeneity could be at the basis of variable effects, the main objective of our study was to evaluate the anti-tumoral activity of a garlic extract in breast cancer cells with a triple negative phenotype. Established triple negative breast cancer (TNBC) cell lines from patient-derived xenografts (PDXs) were used, revealing subtype-dependent effects on morphology, cell cycle, and invasive potential, correlated with the peculiar down-modulation of Akt signaling, a crucial regulator in solid tumors. Our results first demonstrate that the effects of garlic on TNBC breast cancer are not unique and suggest that only more precise knowledge of the mechanisms activated by this natural compound in each tumor will allow for the inclusion of garlic in personalized therapeutic approaches to breast cancer.

Dimeric ferulic acid conjugate as a prodrug for brain targeting after nasal administration of loaded solid lipid microparticles.

OBJECTIVE: Ferulic acid (Fer) displays antioxidant/anti-inflammatory properties useful against neurodegenerative diseases. To increase Fer uptake and its central nervous system residence time, a dimeric prodrug, optimizing the Fer loading on nasally administrable solid lipid microparticles (SLMs), was developed. METHODS: The prodrug was synthesized as Fer dimeric conjugate methylated on the carboxylic moiety. Prodrug antioxidant/anti-inflammatory properties and ability to release Fer in physiologic environments were evaluated. Tristearin or stearic acid SLMs were obtained by hot emulsion technique. In vivo pharmacokinetics were quantified by HPLC. RESULTS: The prodrug was able to release Fer in physiologic environments (whole blood and brain homogenates) and induce in vitro antioxidant/anti-inflammatory effects. Its half-life in rats was 18.0 ± 1.9 min. Stearic acid SLMs, exhibiting the highest prodrug loading and dissolution rate, were selected for nasal administration to rats (1 mg/kg dose), allowing to obtain high prodrug bioavailability and prolonged residence in the cerebrospinal fluid, showing AUC (Area Under Concentration) values (108.5 ± 3.9 µg∙mL-1∙min) up to 30 times over those of Fer free drug, after its intravenous/nasal administration (3.3 ± 0.3/5.16 ± 0.20 µg∙mL-1∙min, respectively) at the same dose. Chitosan presence further improved the prodrug brain uptake. CONCLUSIONS: Nasal administration of prodrug-loaded SLMs can be proposed as a noninvasive approach for neurodegenerative disease therapy.

Influence of Post-Harvest 1-Methylcyclopropene (1-MCP) Treatment and Refrigeration on Chemical Composition, Phenolic Profile and Antioxidant Modifications during Storage of Abate Fétel Pears.

'Abate Fétel', a winter cultivar, is the most important pear cultivar in Italy; its fruits are appreciated by consumers for their aroma, texture and balanced sweet and sour taste. Maintaining high-quality characteristics to prolong the shelf-life of fruit and preserve the sensory and nutritional quality is a priority for the food industry. The aim of our study was to test the effectiveness of 1-methylcyclopropene (1-MCP) and cold storage in prolonging the shelf-life of these fruits, which were harvested at maturity at two different times. This work focused on the effects of different storage treatments and two ripening times on (i) the chemical composition of Abate Fétel pulp fruits to preserve their sweet taste and aroma and (ii) the phenolic profile composition and antioxidant activity of the peel, which is naturally rich in phytochemicals and important for the fruit's shelf-life and in the functional food industry for its high nutritional value. Abate Fétel fruits were harvested at the optimal commercial maturity stage, first on 15 September, having been treated with 1-MCP and stored for 2 months at cold temperatures; the other fruits were harvested at the end of September and stored in a cold cell for 2 months. The fruit pulp was tested for glucose and fructose, pH, acidity and organic acids (malic, citric, fumaric and shikimic), phenolic content and phenolic compounds (chlorogenic and caffeic acids, rutin, hyperoside, kaempferol-3-rutinoside and isoquercitrin), and the antioxidant activities in the fruit peels were measured. Treating the fruits with 1-MCP better preserved the phytochemical compounds compared to simple refrigeration, preserving the fruit's quality and prolonging its shelf-life. All the treatments help to maintain the glucose and fructose content and the acidity, preserving the aroma and organoleptic characteristics.

Future Perspective and Technological Innovation in Cheese Making Using Artichoke (Cynara scolymus) as Vegetable Rennet: A Review.

Milk coagulation is a process used for the formulation of different dairy products such as cheese. In this process, milk undergoes changes in its chemical stability thanks to acidification or enzymatic reactions. Traditionally, milk coagulation has been carried out with rennet of animal origin, but recently, the research of new types of rennet such as microbial rennet and vegetable rennet has increased. This study aims to present an organized review of the most relevant information on lactic coagulation, its relationship with vegetable rennets, and the importance of the botanical genus Cynara in the extraction of vegetable rennets, focusing on the coagulant potential of artichoke (Cynara scolymus). We conducted this literature review and found that lactic coagulation and vegetable rennets are linked through the enzymatic activity of the latter. The results of the main studies demonstrated a strong relationship between vegetable rennets and protease enzymes as well as the presence of these enzymes in extracts of cardoon (Cynara scolymus) and artichoke (Cynara scolymus). In addition, studies highlight the presence of thistle extracts in artisanal cheese preparations in the Iberian Peninsula. Based on the results of the studies, a comparison between cheeses made with vegetable rennet and those made with traditional rennet was also carried out. Although the results show that the use of vegetable rennet in the manufacture of cheese can confer undesirable characteristics, the use of extracts from Cynara plants demonstrates that vegetable rennets have an industrial potential, especially the one obtained from artichoke (Cynara scolymus) due to its high availability. Nevertheless, specific studies are required for a better understanding and application of this rennet.

The Effect of Different Storage Conditions on Phytochemical Composition, Shelf-Life, and Bioactive Compounds of Voghiera Garlic PDO.

Voghiera garlic is an Italian white garlic variety which obtained in 2010 the Protected Designation of Origin. It is widely used for culinary purposes or as an ingredient for supplement production due to its phytochemical compositions. The storage conditions seem to be crucial to retain the high quality of garlic bulbs and their by-products, taking into account the high importance of organosulfur and phenolic compounds for the bioactive potency of garlic and its shelf-life. This study aims to examine the effect of storage on the phytochemical composition, biological effects, and shelf-life of Voghiera garlic PDO. In detail, we considered (i) -4 °C (industrial storage) for 3, 6, and 9 months; (ii) +4 °C for 3 months (home conservation), and (iii) -4 °C for 3 months, plus +4 °C for another 3 months. We focused our attention on the organosulfur compounds, total condensed tannins, flavonoids, phenolic compounds, and related antioxidant activity changes during the storage period. To evaluate the bioactive effects, the Voghiera garlic extracts at different storage conditions were administered to a breast cancer cell line, while antioxidant and anti-inflammatory activity was detected using macrophage RAW 264.7 cells. We observed a decrease in sulfur compounds after 6 months which correlated to a decrease in bioactive effects, while the number of antioxidant compounds was stable during the storage period, showing the good effect of refrigerated temperature in maintaining garlic bulb shelf-life.

Biological activity of new bioactive steroids deriving from biotransformation of cortisone.

Cortisone is a metabolite belonging to the corticosteroid class that is used pharmaceutically directly as a drug or prodrug. In addition to its large consumption, its use is linked to several side effects, so pharmaceutical research aims to develop effective drugs with low or no side effects, alternative compounds to cortisone are part of an active investment in ongoing research on drug discovery. Since biotransformation can be considered a source of new molecules with potential therapeutic use, the present work focuses on a preliminary in vitro study aimed at evaluating the mutagenic, anti-inflammatory, antioxidant and neuroprotective activity of SCA and SCB molecules obtained from the biotransformation of cortisone using Rh. Rhodnii strain DSM 43960. The results obtained are very encouraging due to the safety of biotransformed compounds with reference to genotoxicity checked by Ames test, to the very high antioxidant capacity and to the anti-inflammatory activity. In fact, thecompounds inhibited both the TNFα-stimulated expression and secretion of NFkB target cytokines, and COX activity, and can activate the glucocorticoid receptor. Finally SCA and SCB exhibited neuroprotective properties.

Effects of Microencapsulated Ferulic Acid or Its Prodrug Methyl Ferulate on Neuroinflammation Induced by Muramyl Dipeptide.

Ferulic acid (Fer) is known for its antioxidant and anti-inflammatory activities, which are possibly useful against neurodegenerative diseases. Despite the ability of Fer to permeate the brain, its fast elimination from the body does not allow its therapeutic use to be optimized. The present study proposes the preparation and characterization of tristearin- or stearic acid-based solid lipid microparticles (SLMs) as sustained delivery and targeting systems for Fer. The microparticles were produced by conventional hot emulsion techniques. The synthesis of the methyl ester of Fer (Fer-Me) allowed its encapsulation in the SLMs to increase. Fer-Me was hydrolyzed to Fer in rat whole blood and liver homogenate, evidencing its prodrug behavior. Furthermore, Fer-Me displayed antioxidant and anti-inflammatory properties. The amount of encapsulated Fer-Me was 0.719 ± 0.005% or 1.507 ± 0.014% in tristearin or stearic acid SLMs, respectively. The tristearin SLMs were able to control the prodrug release, while the stearic acid SLMs induced a significant increase of its dissolution rate in water. Jointly, the present results suggest that the tristearin SLMs loaded with Fer-Me could be a potential formulation against peripheral neuropathic pain; conversely, the stearic acid SLMs could be useful for Fer-Me uptake in the brain after nasal administration of the formulation.

Nutrients and Main Secondary Metabolites Characterizing Extracts and Essential Oil from Fruits of Ammodaucus leucotrichus Coss. & Dur. (Western Sahara).

The ethnobotany of the Sahrawi people considers various species of plants and crude drugs as food, cooking spices and traditional health remedies. From among these, the fruits of Ammodaucus leucotrichus Coss. & Dur. (Apiaceae), known as Saharan cumin, were chosen for our research. The present paper reports a proximate composition and mineral element analysis of various samples of A. leucotrichus fruits, collected during the balsamic period (full fruiting) from plants grown in Bir Lehlu (Western Sahara) and purchased in a local market (Tindouf). These analyses pointed out interesting nutritional values of the crude drug. Decoction and alcoholic extract, analyzed by HPLC-DAD, evidenced ammolactone-A and R-perillaldehyde as the two main isolated constituents, particularly in the ethanolic extracts (ammolactone-A, market sample: 51.71 ± 0.39 mg/g dry extract; wild sample: 111.60 ± 1.80 mg/g dry extract; R-perillaldehyde, market sample: 145.95 ± 0.35 mg/g dry extract; wild sample: 221.40 ± 0.30 mg/g dry extract). The essential oils, obtained through hydrodistillation, were characterized by GC-MS and evidenced R-perillaldehyde (market sample: 53.21 ± 1.52%; wild sample: 74.01 ± 1.75%) and limonene (market sample: 35.15 ± 1.68%; wild sample: 19.90 ± 1.86%) as the most abundant compounds. The R configuration of perillaldehyde was ascertained and a complete description of the 1H and 13C NMR spectra of ammolactone-A was performed.

Therapeutic Potential of Allicin and Aged Garlic Extract in Alzheimer's Disease.

Garlic, Allium sativum, has long been utilized for a number of medicinal purposes around the world, and its medical benefits have been well documented. The health benefits of garlic likely arise from a wide variety of components, possibly working synergistically. Garlic and garlic extracts, especially aged garlic extracts (AGEs), are rich in bioactive compounds, with potent anti-inflammatory, antioxidant and neuroprotective activities. In light of these effects, garlic and its components have been examined in experimental models of Alzheimer's disease (AD), the most common form of dementia without therapy, and a growing health concern in aging societies. With the aim of offering an updated overview, this paper reviews the chemical composition, metabolism and bioavailability of garlic bioactive compounds. In addition, it provides an overview of signaling mechanisms triggered by garlic derivatives, with a focus on allicin and AGE, to improve learning and memory.

Antioxidant Activity of Resveratrol Diastereomeric Forms Assayed in Fluorescent-Engineered Human Keratinocytes.

Resveratrol is a powerful antioxidant molecule. In the human diet, its most important source is in Vitis vinifera grape peel and leaves. Resveratrol exists in two isoforms, cis- and trans. The diastereomeric forms of many drugs have been reported as affecting their activity. The aim of this study was to set up a cellular model to investigate how far resveratrol could counteract cytotoxicity in an oxidant agent. For this purpose, a keratinocyte cell line, which was genetically engineered with jelly fish green fluorescent protein, was treated with the free radical promoter Cumene hydroperoxide. The antioxidant activity of the trans-resveratrol and its diastereomeric mixture was evaluated indirectly in these treated fluorescent-engineered keratinocytes by analyzing the cell number and cell proliferation index. Our results demonstrate that cells, which were pre-incubated with resveratrol, reverted the oxidative damage progression induced by this free radical agent. In conclusion, fluorescent-engineered human keratinocytes represent a rapid and low-cost cellular model to determine cell numbers by studying emitted fluorescence. Comparative studies carried out with fluorescent keratinocytes indicate that trans-resveratrol is more efficient than diastereomeric mixtures in protecting cells from the oxidative stress.

Anticancer Activity of Aqueous Extracts from Asparagus officinalis L. Byproduct on Breast Cancer Cells.

Cultivation of asparagus (Asparagus officinalis L.; Asp) for food and medicinal use has taken place since the early Roman Empire. Today, Asp represents a worldwide diffuse perennial crop. Lower portions of the spears represent a food industry waste product that can be used to extract bioactive molecules. In this study, aqueous extracts derived from the non-edible portion of the plant (hard stem) were prepared and characterized for chemical content. Furthermore, the biocompatibility and bioactivity of Asp aqueous extracts were assessed in vitro on normal fibroblasts and on breast cancer cell lines. Results showed no interference with fibroblast viability, while a remarkable cytostatic concentration-dependent activity, with significant G1/S cell cycle arrest, was specifically observed in breast cancer cells without apoptosis induction. Asp extracts were also shown to significantly inhibit cell migration. Further analyses showed that Asp extracts were characterized by specific pro-oxidant activity against tumoral cells, and, importantly, that their combination with menadione resulted in a significant enhancement of oxidants production with respect to menadione alone in breast cancer cells but not in normal cells. This selectivity of action on tumoral cells, together with the easiness of their preparation, makes the aqueous Asp extracts very attractive for further investigation in breast cancer research, particularly to investigate their role as possible co-adjuvant agents of clinical drug therapies.

Antioxidant and Antiinflammatory Effects of Epilobium parviflorum, Melilotus officinalis and Cardiospermum halicacabum Plant Extracts in Macrophage and Microglial Cells.

BACKGROUND: We investigated the phenolic content characterizing different plant extracts from Epilobium parviflorum, Cardiospermum halicacabum, and Melilotus officinalis, their antioxidant, antiinflammatory effects, and their mechanism of action. METHODS: plant samples were macerated in 40% ethanol or hot/ cold glycerate and assessed for polyphenols content. The antioxidant activity was investigated by DPPH radical scavenging assay and H2DCFDA test in LPS-stimulated RAW264.7 macrophages and N9 microglial cells. MTS experiments and antiinflammatory properties verified cellular toxicity through NO assay. Interaction with A2A adenosine receptors was evaluated through binding assays using [3H]ZM241385 radioligand. RESULTS: Polyphenols were present in 40% ethanol plant extract, which at 0.1-10 µg/µL achieved good antioxidant effects, with a DPPH radical scavenging rate of about 90%. In LPS-stimulated cells, these plant extracts, at 1µg/µL, did not affect cell vitality, displayed significant inhibition of H2DCFDA and NO production, and inhibited ZM 241385 binding in CHO cells transfected with A2A receptors. RAW 264.7 and N9 cells presented a density of them quantified in 60 ± 9 and 45 ± 5 fmol/mg of protein, respectively. CONCLUSION: Epilobium parviflorum, Cardiospermum halicacabum, and Melilotus officinalis extracts may be considered a source of agents for treating disorders related to oxidative stress and inflammation.

The role of pumpkin pulp extract carotenoids against mycotoxin damage in the blood brain barrier in vitro.

Some mycotoxins such as beauvericin (BEA), ochratoxin A (OTA), and zearalenone (ZEA) can cross the blood brain barrier, which is why we tested the anti-inflammatory action of a pumpkin carotenoid extract (from the pulp) against these mycotoxins and their combinations (OTA+ZEA and OTA+ZEA+BEA) on a blood brain barrier model with co-cultured ECV304 and C6 cells using an untargeted metabolomic approach. The cells were added with mycotoxins at a concentration of 100 nmol/L per mycotoxin and pumpkin carotenoid extract at 500 nmol/L. For control we used only vehicle solvent (cell control) or vehicle solvent with pumpkin extract (extract control). After two hours of exposure, samples were analysed with HPLC-ESI-QTOF-MS. Metabolites were identified against the Metlin database. The proinflammatory arachidonic acid metabolite eoxin (14,15-LTE4) showed lower abundance in ZEA and BEA+OTA+ZEA-treated cultures that also received the pumpkin extract than in cultures that were not treated with the extract. Another marker of inflammation, prostaglandin D2-glycerol ester, was only found in cultures treated with OTA+ZEA and BEA+OTA+ZEA but not in the ones that were also treated with the pumpkin extract. Furthermore, the concentration of the pumpkin extract metabolite dihydromorelloflavone significantly decreased in the presence of mycotoxins. In conclusion, the pumpkin extract showed protective activity against cellular inflammation triggered by mycotoxins thanks to the properties pertinent to flavonoids contained in the pulp.

Ethanol-based garlic extract prevents malignant evolution of non-invasive breast tumor cells induced by moderate hypoxia.

BACKGROUND: In breast cancer, low oxygen availability is associated with a more aggressive phenotype and with malignant evolution of non-invasive cells. Natural compounds have long attracted attention in cancer treatment, and in recent years garlic (Allium sativum) organosulfur derivatives have been shown to negatively affect growth and invasion of tumor cells. METHODS: Homemade ethanol-based garlic extract (GE) was administered to MCF7 and MCF10DCIS breast tumor cell lines grown under moderate hypoxia. Cell cycle, epithelial-to-mesenchymal transition and cancer stem cell markers were evaluated. RESULTS: We revealed that, in the non-invasive MCF10DCIS cells but not in the post-EMT MCF7 cells, low oxygen availability induced the decrease of E-cadherin and the increase of vimentin and motility, that were prevented by GE administration. In both cell lines, treatment with GE counteracted the up-modulation of CD133 positive cells induced by hypoxia. CONCLUSIONS: Overall, our data firstly revealed anti-cancer properties of garlic in non-invasive breast cancer cells. In particular, they demonstrated a protective role of this natural product against the hypoxia-induced increase of molecules that play crucial roles in tumor evolution, suggesting that garlic derivatives can be considered in new approaches for preventing progression of breast tumors from non-invasive to infiltrating lesions.

Nutrient Composition and Antioxidant Performances of Bread-Making Products Enriched with Stinging Nettle (Urtica dioica) Leaves.

Stinging nettle (Urtica dioica) is an edible plant, well-known for its nutritional and nutraceutical properties. Stinging nettle leaves are typically rich in fibers, minerals and vitamins, as well as antioxidant compounds, i.e., polyphenols and carotenoids. Due to these reasons, since ancient times stinging nettle has been widely used in Italy as an ingredient in foods and beverages as a therapeutic agent. This work provides an investigation focused on bread enrichment with nettle leaves and the improvement of bread proximate composition in minerals, fibers and antioxidant compounds during product preparation. The comparison between plain and nettle enriched white bread shows a significant increase in fibers and nutrients, i.e., calcium and copper levels. Nettle enrichment also provides an increase in lutein and ß-carotene, as well as in total phenols and antioxidant activity. These last two nutritional elements are remarkably high in enriched bread and it has been found that phenolic concentration increases during breadmaking steps, from kneading to primary dough fermentation and from secondary fermentation of shaped loaves to baking.

Cytoprotection assessment against mycotoxins on HepG2 cells by extracts from Allium sativum L.

Cytoprotection effects of Allium sativum L garlic extract from a local garlic ecotype from Ferrara (Italy) on hepatocarcinoma cells, HepG2 cells, is presented in this study. This garlic type is known as Voghiera garlic and has been characterized as PDO (Protected designation of Origin) product. Voghiera garlic extract (VGE) was evaluated against beauvericin (BEA) and two zearalenone (ZEA) metabolites (α-zearalenol (α-ZEL) and ß-zearalenol (ß-ZEL))-induced cytotoxicity on HepG2 cells by the MTT (3-4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, over 24 h and 48 h. Direct treatment, simultaneous treatment and pre-treatment strategies at the dilution 1:16-1:00 for VGE and at the concentration range from 0.08 to 2.5 µM for BEA and from 1.6 to 50 µM for both α-ZEL and ß-ZEL were tested. Individual IC50 values were detected at all times assayed for BEA (>0.75 µM) and VGE (dilution upper 1:8) while this was not observed for ZEA's metabolites. When simultaneous strategy of VGE + mycotoxin was tested, cytoprotection with increases of viability (upper 50%) were observed. Lastly, in pre-treatment strategy with VGE, viability of HepG2 cells was significantly protected when α-ZEL was tested. As a result, the greatest cytoprotective effect of VGE in HepG2 cells is obtained when simultaneous treatment strategy was performed.

Isolation, Identification and Investigation of Fermentative Bacteria from Sea Bass (Dicentrarchus labrax): Evaluation of Antifungal Activity of Fermented Fish Meat and By-Products Broths.

During fish production processes, great amounts of by-products are generated, representing ≈30-70% of the initial weight. Thus, this research study is investigating 30 lactic acid bacteria (LAB) derived from the sea bass gastrointestinal tract, for anti-fungal activity. It has been previously suggested that LAB showing high proteolitic activity are the most suitable candidates for such an investigation. The isolation was made using a MRS (Man Rogosa Sharpe) broth cultivation medium at 37 ºC under anaerobiosis conditions, while the evaluation of the enzymatic activity was made using the API® ZYM kit. Taking into account the selected bacteria, a growing research was made fermenting two kinds of broths: (i) by-products (WB), and (ii) meat (MB). Both were fermented at three different times (24, 48 and 72 h). Then, the antifungal activities of both fermented by-products and meat broths were determined qualitatively and quantitatively in solid and liquid medium against two different strains of the genera Penicillium, Aspergillus and Fusarium. After the experiments, a total of 30 colonies were isolated, observing a proteolytic activity in 7 of the isolated strains, which belong to Lactobacillus genus, and the two more active strains were identified by polymerase chain reaction (PCR) as L. plantarum. Several strains evidenced antifungal activity showing an inhibition halo and Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) values between 1-32 g/L and 8-32 g/L, respectively. In conclusion, the isolated bacteria of sea bass had the ability to promote the antifungal activity after the fermentation process, thus being a useful tool to give an added value to fish industry by-products.

Preliminary multi analytical approach to address geographic traceability at the intraspecific level in Scombridae family.

Globalization of seafood product marketing caused the increase of request of an effective fish traceability that enhances the consumer confidence in food safety. In this study, an integrated multi analytical approach based on two different and independent analytical techniques (carbon and nitrogen stable isotopes and fatty acids analysis) was applied in order to identify different fish species and trace their geographical provenience. The investigation was focused on four species (Thunnus thynnus, Thunnus alalunga, Auxis rochei and Scomber scombrus) belonging to the Scombridae family. The DNA barcoding method confirmed genus and species for S. scombrus and A. rochei, but only genus for T. alalunga and T. thynnus. Carbon and nitrogen stable isotopes results evidenced different fish diets and trophic positions, whereas fatty acids analysis displayed that the unsaturated prevailed (∼60 %) over the saturated compounds with a variation among the species and the geographical area in particular for docosahexaenoic and eicosapentaenoic acids percentage. The principal component analysis applied to stable isotopes and fatty acids evidenced a good discrimination among species and their geographical catching area. This multi-disciplinary analytical approach could represent a promising tool to identify the commercial fish and trace their origin in order to guarantee the health of consumers.

Impact of Fermentation on the Recovery of Antioxidant Bioactive Compounds from Sea Bass Byproducts.

The aim of the present research was to obtain antioxidant compounds through the fermentation of fish byproducts by bacteria isolated from sea bass viscera. To that purpose, bacteria from sea bass stomach, intestine, and colon were isolated. With the selected bacteria, growing research was undertaken, fermenting different broths prepared with sea bass meat or byproducts. After the fermentation, the antioxidant activity, phenolic acids, and some proteins were evaluated. The main phenolic acids obtained were DL-3-phenyl-lactic acid and benzoic acid at a maximum concentration of 466 and 314 ppb, respectively. The best antioxidant activity was found in the extracts obtained after the fermentation of fish byproducts broth by bacteria isolated from the colon (6502 µM TE) and stomach (4797 µM TE). Moreover, a positive correlation was found between phenolic acids obtained after the fermentation process and the antioxidant activity of the samples. It was also concluded that the lactic acid bacteria isolated from sea bass had an important proteolytic capacity and were able to synthesize phenolic acids with antioxidant capacity. This work has shown the relevance of fermentation as a useful tool to valorize fish byproducts, giving them an added economic value and reducing their environmental impact.

HPLC-UV/Vis-APCI-MS/MS Determination of Major Carotenoids and Their Bioaccessibility from "Delica" (Cucurbita maxima) and "Violina" (Cucurbita moschata) Pumpkins as Food Traceability Markers.

Carotenoids are a widespread group of fat-soluble pigments, and their major nutritional importance comes from their pro-vitamin A activity and their antioxidant capacity. In this study, two different pumpkin cultivars (Cucurbita maxima, also named `Delica' and Cucurbita moschata, also known as `Violina') from the southern Po Delta area were investigated in terms of carotenoid content and the influence of food processing on compositional changes and carotenoid bioaccessibility. Quali- and quantitative determination of carotenoids in sample extracts were performed on a C30 column by means of an online coupled HPLC-UV/Vis-APCI-MS/MS technique. The identification of separated compounds was tentatively achieved by merging (i) chromatographic data, (ii) UV-Vis spectra, and (iii) MS/MS fragmentation spectra. The chromatographic profiles for the two cultivars showed qualitative differences. Two major carotenoids were considered for quantification purposes and further investigations: lutein and ß -carotene. Quantification of target carotenoids was performed with external calibration through analytical standards. The concentration of lutein and ß -carotene was higher in C. maxima than in the other variety, C. moschata. Carotenoids are susceptible to degradation (isomerization and oxidation) during food processing (i.e., cooking), and the concentration of lutein and ß -carotene were monitored in oven-cooked and steam-cooked pumpkins. The steam-cooking process was superior in terms of limiting carotenoid loss. A complete functional profile of pumpkins as a source of carotenoids was gained with the evaluation of their in vitro bioaccessibility and their bioavailability after intake during human digestion. Bioaccessibility of lutein and ß -carotene were estimated by an in vitro static digestion model that involved salivary, gastric, and duodenal phases. Bioaccessibility values progressively increased from the salivary to the duodenal phase for both pumpkin varieties and cooking methods. Bioaccessibility of lutein was always lower than ß -carotene for both cultivars and for both cooking methods. Bioaccessibility values for lutein and ß -carotene changed from 1.93% to 2.34% vs. 4.94% and 8.83% in the salivary phase, from 2.7% to 4.63% vs. 7.83% and 15.60% in the gastric phase, and from 10.04% to 13.42% vs. 25.81% and 35.32% in the duodenal phase. For both target compounds, bioaccessibility in the duodenal phase was more than twice the gastric values, and it underlined that the type of cooking did not influence release from the initial matrix.