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SERENA MARTINI

CULTORE DELLA MATERIA
Dipartimento di Scienze della Vita sede ex-Agraria

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Pubblicazioni

2023 - Lung UltrasouNd Guided surfactant therapy in preterm infants: an international multicenter randomized control trial (LUNG study) [Articolo su rivista]
Corsini, I.; Rodriguez-Fanjul, J.; Raimondi, F.; Boni, L.; Berardi, A.; Aldecoa-Bilbao, V.; Alonso-Ojembarrena, A.; Ancora, G.; Aversa, S.; Beghini, R.; Meseguer, N. B.; Capasso, L.; Chesi, F.; Ciarcia, M.; Concheiro, A.; Corvaglia, L.; Ficial, B.; Filippi, L.; Carballal, J. F.; Fusco, M.; Gatto, S.; Ginovart, G.; Gregorio-Hernandez, R.; Lista, G.; Sanchez-Luna, M.; Martini, S.; Massenzi, L.; Miselli, F.; Mercadante, D.; Mosca, F.; Palacio, M. T.; Perri, A.; Piano, F.; Prieto, M. P.; Fernandez, L. R.; Risso, F. M.; Savoia, M.; Staffler, A.; Vento, G.; Dani, C.
abstract

Background: The management of respiratory distress syndrome (RDS) in premature newborns is based on different types of non-invasive respiratory support and on surfactant replacement therapy (SRT) to avoid mechanical ventilation as it may eventually result in lung damage. European guidelines currently recommend SRT only when the fraction of inspired oxygen (FiO2) exceeds 0.30. The literature describes that early SRT decreases the risk of bronchopulmonary dysplasia (BPD) and mortality. Lung ultrasound score (LUS) in preterm infants affected by RDS has proven to be able to predict the need for SRT and different single-center studies have shown that LUS may increase the proportion of infants that received early SRT. Therefore, the aim of this study is to determine if the use of LUS as a decision tool for SRT in preterm infants affected by RDS allows for the reduction of the incidence of BPD or death in the study group. Methods/design: In this study, 668 spontaneously-breathing preterm infants, born at 25+0 to 29+6 weeks’ gestation, in nasal continuous positive airway pressure (nCPAP) will be randomized to receive SRT only when the FiO2 cut-off exceeds 0.3 (control group) or if the LUS score is higher than 8 or the FiO2 requirements exceed 0.3 (study group) (334 infants per arm). The primary outcome will be the difference in proportion of infants with BPD or death in the study group managed compared to the control group. Discussion: Based on previous published studies, it seems that LUS may decrease the time to administer surfactant therapy. It is known that early surfactant administration decreases BPD and mortality. Therefore, there is rationale for hypothesizing a reduction in BPD or death in the group of patients in which the decision to administer exogenous surfactant is based on lung ultrasound scores. Trial registration: ClinicalTrials.gov identifier NCT05198375 . Registered on 20 January 2022.

2023 - Red-skinned onion phenolic compounds stability and bioaccessibility: A comparative study between deep-frying and air-frying [Articolo su rivista]
Cattivelli, A.; Di Lorenzo, A.; Conte, A.; Martini, S.; Tagliazucchi, D.
abstract

In this study, the effect of deep-frying and air-frying on the stability and gastro-intestinal release of red-skinned onion phenolic compounds have been assessed by high-resolution mass spectrometry. Forty-four phenolic compounds were identified and quantified in raw onion. Both the frying treatments caused an increase in total phenolic compounds, which was more evident after air-frying (47.4% vs 18.6% increase). This increase was mainly a consequence of the water loss observed during frying. Quercetin was the most important phenolic compound detected both in raw and air-fried samples (99.5 ± 1.7 and 133 ± 2 μmol/100 g, respectively) while deep-fried onion was richer in quercetin-4’-O-glucoside and quercetin-3-O-glucoside-4'-O-glucoside (48.7 ± 0.9 and 41.8 ± 0.3 μmol/100 g, respectively). After digestion, 38.6%, 60.5% and 89.6% of total phenolic compounds were bioaccessible in raw, deep-fried and air-fried onion. Air-fried onion exhibited the highest concentration of bioaccessible phenolic compounds (206 ± 1 μmol/100 g). Oxidative degradation and hydrolysis reactions of quercetin-hexosides occurred during in vitro digestion. Hence, air-frying can be considered a healthy cooking method able to preserve and release after digestion most of the health-promoting compounds found in raw onion and with a lower amount of fats and polar toxic compounds compared to deep-frying.

2023 - The Italian data on SARS-CoV-2 infection in transplanted patients support an organ specific immune response in liver recipients [Articolo su rivista]
Rendina, M.; Barone, M.; Lillo, C.; Trapani, S.; Masiero, L.; Trerotoli, P.; Puoti, F.; Lupo, L. G.; Tandoi, F.; Agnes, S.; Grieco, A.; Andorno, E.; Marenco, S.; Giannini, E. G.; Baccarani, U.; Toniutto, P.; Carraro, A.; Colecchia, A.; Cescon, M.; Morelli, M. C.; Cillo, U.; Burra, P.; Angeli, P.; Colledan, M.; Fagiuoli, S.; De Carlis, L.; Belli, L.; De Simone, P.; Carrai, P.; Di Benedetto, F.; De Maria, N.; Ettorre, G. M.; Giannelli, V.; Gruttadauria, S.; Volpes, R.; Corsale, S.; Mazzaferro, V.; Bhoori, S.; Romagnoli, R.; Martini, S.; Rossi, G.; Caccamo, L.; Donato, M. F.; Rossi, M.; Ginanni Corradini, S.; Spada, M.; Maggiore, G.; Tisone, G.; Lenci, I.; Vennarecci, G.; Tortora, R.; Vivarelli, M.; Svegliati Baroni, G.; Zamboni, F.; Mameli, L.; Tafuri, S.; Simone, S.; Gesualdo, L.; Cardillo, M.; Di Leo, A.
abstract

Introduction: The study of immune response to SARSCoV-2 infection in different solid organ transplant settings represents an opportunity for clarifying the interplay between SARS-CoV-2 and the immune system. In our nationwide registry study from Italy, we specifically evaluated, during the first wave pandemic, i.e., in non-vaccinated patients, COVID-19 prevalence of infection, mortality, and lethality in liver transplant recipients (LTRs), using non-liver solid transplant recipients (NL-SOTRs) and the Italian general population (GP) as comparators. Methods: Case collection started from February 21 to June 22, 2020, using the data from the National Institute of Health and National Transplant Center, whereas the data analysis was performed on September 30, 2020.To compare the sex- and age-adjusted distribution of infection, mortality, and lethality in LTRs, NL-SOTRs, and Italian GP we applied an indirect standardization method to determine the standardized rate. Results: Among the 43,983 Italian SOTRs with a functioning graft, LTRs accounted for 14,168 patients, of whom 89 were SARS-CoV-2 infected. In the 29,815 NL-SOTRs, 361 cases of SARS-CoV-2 infection were observed. The geographical distribution of the disease was highly variable across the different Italian regions. The standardized rate of infection, mortality, and lethality rates in LTRs resulted lower compared to NL-SOTRs [1.02 (95%CI 0.81-1.23) vs. 2.01 (95%CI 1.8-2.2); 1.0 (95%CI 0.5-1.5) vs. 4.5 (95%CI 3.6-5.3); 1.6 (95%CI 0.7-2.4) vs. 2.8 (95%CI 2.2-3.3), respectively] and comparable to the Italian GP. Discussion: According to the most recent studies on SOTRs and SARS-CoV-2 infection, our data strongly suggest that, in contrast to what was observed in NL-SOTRs receiving a similar immunosuppressive therapy, LTRs have the same risk of SARS-CoV-2 infection, mortality, and lethality observed in the general population. These results suggest an immune response to SARS-CoV-2 infection in LTRS that is different from NL-SOTRs, probably related to the ability of the grafted liver to induce immunotolerance.

2022 - Application of a Combined Peptidomics and In Silico Approach for the Identification of Novel Dipeptidyl Peptidase-IV-Inhibitory Peptides in In Vitro Digested Pinto Bean Protein Extract [Articolo su rivista]
Martini, S.; Cattivelli, A.; Conte, A.; Tagliazucchi, D.
abstract

The conventional approach in bioactive peptides discovery, which includes extensive bioassay-guided fractionation and purification processes, is tedious, time-consuming and not always successful. The recently developed bioinformatics-driven in silico approach is rapid and cost-effective; however, it lacks an actual physiological significance. In this study a new integrated peptidomics and in silico method, which combines the advantages of the conventional and in silico approaches by using the pool of peptides identified in a food hydrolysate as the starting point for subsequent application of selected bioinformatics tools, has been developed. Pinto bean protein extract was in vitro digested and peptides were identified by peptidomics. The pool of obtained peptides was screened by in silico analysis and structure–activity relationship modelling. Three peptides (SIPR, SAPI and FVPH) were selected as potential inhibitors of the dipeptidyl-peptidase-IV (DPP-IV) enzyme by this integrated approach. In vitro bioactivity assay showed that all three peptides were able to inhibit DPP-IV with the tetra-peptide SAPI showing the highest activity (IC50 = 57.7 µmol/L). Indeed, a new possible characteristic of peptides (i.e., the presence of an S residue at the N-terminus) able to inhibit DPP-IV was proposed.

2022 - Cooking and In Vitro Digestion Modulate the Anti-Diabetic Properties of Red-Skinned Onion and Dark Purple Eggplant Phenolic Compounds [Articolo su rivista]
Cattivelli, A.; Conte, A.; Martini, S.; Tagliazucchi, D.
abstract

The intake of phenolic-rich foods is an emerging preventive approach for the management of type 2 diabetes, thanks to the ability of these compounds to inhibit some key metabolic enzymes. In this study, the influence of cooking and in vitro digestion on the α-glucosidase, α-amylase, and dipeptidyl-peptidase IV (DPP-IV) inhibitory activity of red-skinned onion (RSO) and dark purple eggplant (DPE) phenolic fractions was assessed. The applied cooking procedures had different influences on the total and individual phenolic compounds gastrointestinal bioaccessibility. DPE in vitro digested phenolic fractions displayed no inhibitory activity versus α-amylase and DPP-IV, whereas the fried DPE sample exhibited moderate inhibitory activity against α-glucosidase. This sample mainly contained hydroxycinnamic acid amides that can be responsible for the observed effect. Contrariwise, raw and cooked in vitro digested RSO phenolic fractions inhibited all three enzymes but with different effectiveness. Fried and raw RSO samples were the most active against them. Statistical analysis pointed out that quercetin mono-hexosides (mainly quercetin-4′-O-hexoside) were responsible for the inhibition of α-glucosidase, whereas quercetin dihexosides (mainly quercetin-3-O-hexoside-4′-O-hexoside) were responsible for the DPP-IV-inhibitory activity of RSO samples. An accurate design of the cooking methods could be essential to maximize the release of individual phenolic compounds and the related bioactivities.

2022 - Metabolomic and Transcriptional Profiling of Oleuropein Bioconversion into Hydroxytyrosol during Table Olive Fermentation by Lactiplantibacillus plantarum [Articolo su rivista]
Vaccalluzzo, A; Solieri, L; Tagliazucchi, D; Cattivelli, A; Martini, S; Pino, A; Caggia, C; Randazzo, Cl
abstract

2021 - An integrated peptidomics and in silico approach to identify novel anti-diabetic peptides in parmigiano-reggiano cheese [Articolo su rivista]
Martini, S.; Solieri, L.; Cattivelli, A.; Pizzamiglio, V.; Tagliazucchi, D.
abstract

Inhibition of key metabolic enzymes linked to type-2-diabetes (T2D) by food-derived compounds is a preventive emerging strategy in the management of T2D. Here, the impact of Parmigiano- Reggiano (PR) cheese peptide fractions, at four different ripening times (12, 18, 24, and 30 months), on the enzymatic activity of α-glucosidase, α-amylase, and dipeptidyl peptidase-IV (DPPIV) as well as on the formation of fluorescent advanced glycation end-products (fAGEs) was assessed. The PR peptide fractions were able to inhibit the selected enzymes and fAGEs formation. The 12-month-ripening PR sample was the most active against the three enzymes and fAGEs. Mass spectrometry analysis enabled the identification of 415 unique peptides, 54.9% of them common to the four PR samples. Forty-nine previously identified bioactive peptides were found, mostly characterized as angiotensin-converting enzyme-inhibitors. The application of an integrated approach that combined peptidomics, in silico analysis, and a structure–activity relationship led to an efficient selection of 6 peptides with potential DPP-IV and α-glucosidase inhibitory activities. Peptide APFPE was identified as a potent novel DPP-IV inhibitor (IC50 = 49.5 ± 0.5 μmol/L). In addition, the well-known anti-hypertensive tripeptide, IPP, was the only one able to inhibit the three digestive enzymes, highlighting its possible new and pivotal role in diabetes management.

2021 - Antioxidant and Angiotensin I-Converting Enzyme (ACE) Inhibitory Peptides Obtained from Alcalase Protein Hydrolysate Fractions of Hemp (Cannabis sativa L.) Bran [Articolo su rivista]
Samaei, S. P.; Martini, S.; Tagliazucchi, D.; Gianotti, A.; Babini, E.
abstract

Proteins from hemp bran (HPB), a byproduct of the hemp seed food-processing chain, were chemically extracted, hydrolyzed by Alcalase, and separated by membrane ultrafiltration into four fractions (MW <1, 1-3, 3-5, and >5 kDa). The antioxidant and antihypertensive properties of the initial extract and the fractions were evaluated by in vitro assays for their ability to scavenge radical species, bind with metal ions, reduce ferric ions, and inhibit angiotensin-converting enzyme (ACE) activity. Bioactive peptides were identified by high-resolution mass spectrometry and sequence comparison with BIOPEP and BioPep DB databases. The hydrolysate was strongly antioxidant and ACE-inhibiting; the most bioactive peptides were further concentrated by ultrafiltration. Of the 239 peptides identified, 47 (12 antioxidant and 35 ACE-inhibitory) exhibited structural features correlated with the specific bioactivity. These results highlight the promise of hydrolysate and size-based HPB fractions as natural functional ingredients for the food or pharmaceutical industry.

2021 - Black, green, and pink pepper affect differently lipid oxidation during cooking and in vitro digestion of meat [Articolo su rivista]
Martini, S.; Cattivelli, A.; Conte, A.; Tagliazucchi, D.
abstract

Lipid oxidation products generated during meat digestion may contribute to the apparent epidemiological link between red meat intake and the risk of cardiovascular diseases and colorectal cancer. The aim of this work was to assess the lipid oxidation inhibitory activity of black, green, and pink pepper during cooking and in vitro digestion of meat. Peppers were characterized for their phenolic profiles by LC-ESI-MS and the antioxidant properties. Pink pepper showed the highest phenolic content and antioxidant activities. Then, the peppers were added to meat either before or after cooking, and the meat was subjected to in vitro digestion. Pink pepper added before cooking was the most effective, with an inhibition of 80% and 72% in lipid hydroperoxides and TBA-RS formation after digestion, respectively. These findings suggest that peppers, particularly pink pepper, can be used to minimize lipid oxidation in the gastro-intestinal tract and for the design of healthy dietary patterns.

2021 - Characterization of yeasts isolated from parmigiano reggiano cheese natural whey starter: From spoilage agents to potential cell factories for whey valorization [Articolo su rivista]
Martini, S.; Bonazzi, M.; Malorgio, I.; Pizzamiglio, V.; Tagliazucchi, D.; Solieri, L.
abstract

Whey is the main byproduct of the dairy industry and contains sugars (lactose) and proteins (especially serum proteins and, at lesser extent, residual caseins), which can be valorized by the fermentative action of yeasts. In the present study, we characterized the spoilage yeast population inhabiting natural whey starter (NWS), the undefined starter culture of thermophilic lactic acid bacteria used in Parmigiano Reggiano (PR) cheesemaking, and evaluated thermotolerance, mating type, and the aptitude to produce ethanol and bioactive peptides from whey lactose and proteins, respectively, in a selected pool of strains. PCR-RFLP assay of ribosomal ITS regions and phylogenetic analysis of 26S rDNA D1/D2 domains showed that PR NWS yeast population consists of the well-documented Kluyveromyces marxianus, as well as of other species (Saccharomyces cerevisiae, Wickerhamiella pararugosa, and Torulaspora delbrueckii), with multiple biotypes scored within each species as demonstrated by (GTG)5-based MSP-PCR. Haploid and diploid K. marxianus strains were identified through MAT genotyping, while thermotolerance assay allowed the selection of strains suitable to grow up to 48◦C. In whey fermentation trials, one thermotolerant strain was suitable to release ethanol with a fermentation efficiency of 86.5%, while another candidate was able to produce the highest amounts of both ethanol and bioactive peptides with potentially anti-hypertensive function. The present work demonstrated that PR NWS is a reservoir of ethanol and bioactive peptides producer yeasts, which can be exploited to valorize whey, in agreement with the principles of circularity and sustainability.

2021 - Clinical features and comorbidity pattern of HCV infected migrants compared to native patients in care in Italy: A real-life evaluation of the PITER cohort [Articolo su rivista]
Quaranta, M. G.; Ferrigno, L.; Tata, X.; D'Angelo, F.; Massari, M.; Coppola, C.; Biliotti, E.; Giorgini, A.; Laccabue, D.; Ciancio, A.; Blanc, P. L.; Margotti, M.; Ieluzzi, D.; Brunetto, M. R.; Barbaro, F.; Russo, F. P.; Beretta, I.; Morsica, G.; Verucchi, G.; Saracino, A.; Galli, M.; Kondili, L. A.; Mazzaro, C.; Bertola, M.; Benedetti, A.; Schiada, L.; Cucco, M.; Giacometti, A.; Brescini, L.; Castelletti, S.; Fiorentini, A.; Angarano, G.; Milella, M.; Leo, A. D.; Rendina, M.; Salvatore D'ABRAMO, F.; Lillo, C.; Iannone, A.; Piazzolla, M.; Badia, L.; Piscaglia, F.; Benevento, F.; Serio, I.; Castelli, F.; Zaltron, S.; Spinetti, A.; Odolini, S.; Bruno, R.; Mondelli, M.; Chessa, L.; Loi, M.; Torti, C.; Costa, C.; Mazzitelli, M.; Pisani, V.; Scaglione, V.; Trecarichi, E. M.; Zignego, A. L.; Monti, M.; Madia, F.; Attala, L.; Pierotti, P.; Salomoni, E.; Mariabelli, E.; Santantonio, T. A.; Bruno, S. R.; Cela, E. M.; Bassetti, M.; Mazzarello, G.; Alessandrini, A. I.; Biagio, A. D.; Nicolini, L. A.; Raimondo, G.; Filomia, R.; Aghemo, A.; Meli, R.; Lazzarin, A.; Salpietro, S.; Fracanzani, A. L.; Fatta, E.; Lombardi, R.; Lampertico, P.; Borghi, M.; D'Ambrosio, R.; Degasperi, E.; Puoti, M.; Baiguera, C.; D'Amico, F.; Vinci, M.; Rumi, M. G.; Zuin, M.; Zermiani, P.; Andreone, P.; Caraceni, P.; Guarneri, V.; Villa, E.; Bernabucci, V.; Bristot, L.; Paradiso, M. L.; Migliorino, G.; Gambaro, A.; Lapadula, G.; Spolti, A.; Soria, A.; Invernizzi, P.; Ciaccio, A.; Luca, M.; Malinverno, F.; Ratti, L.; Amoruso, D. C.; Pisano, F.; Scarano, F.; Staiano, L.; Morisco, F.; Cossiga, V.; Gentile, I.; Buonomo, A. R.; Foggia, M.; Zappulo, E.; Federico, A.; Dallio, M.; Coppola, N.; Sagnelli, C.; Martini, S.; Monari, C.; Nardone, G.; Sgamato, C.; Chemello, L.; Cavalletto, L.; Sterrantino, D.; Zanetto, A.; Zanaga, P.; Brancaccio, G.; Craxi, A.; Petta, S.; Calvaruso, V.; Crapanzano, L.; Madonia, S.; Cannizzaro, M.; Bruno, E. M.; Licata, A.; Amodeo, S.; Capitano, A. R.; Ferrari, C.; Negri, E.; Orlandini, A.; Pesci, M.; Gulminetti, R.; Pagnucco, L.; Parruti, G.; Stefano, P. D.; Coco, B.; Corsini, R.; Garlassi, E.; Andreoni, M.; Teti, E.; Cerva, C.; Baiocchi, L.; Grassi, G.; Gasbarrini, A.; Pompili, M.; Siena, M. D.; Taliani, G.; Spaziante, M.; Persico, M.; Masarone, M.; Aglitti, A.; Calvanese, G.; Anselmo, M.; Leo, P. D.; Marturano, M.; Saracco, G. M.
abstract

Background: Direct-acting antivirals are highly effective for the treatment of hepatitis C virus (HCV) infection, regardless race/ethnicity. We aimed to evaluate demographic, virological and clinical data of HCV-infected migrants vs. natives consecutively enrolled in the PITER cohort. Methods: Migrants were defined by country of birth and nationality that was different from Italy. Mann-Whitney U test, Chi-squared test and multiple logistic regression were used. Results: Of 10,669 enrolled patients, 301 (2.8%) were migrants: median age 47 vs. 62 years, (p < 0.001), females 56.5% vs. 45.3%, (p < 0.001), HBsAg positivity 3.8% vs. 1.4%, (p < 0.05). Genotype 1b was prevalent in both groups, whereas genotype 4 was more prevalent in migrants (p < 0.05). Liver disease severity and sustained virologic response (SVR) were similar. A higher prevalence of comorbidities was reported for natives compared to migrants (p < 0.05). Liver disease progression cofactors (HBsAg, HIV coinfection, alcohol abuse, potential metabolic syndrome) were present in 39.1% and 47.1% (p > 0.05) of migrants and natives who eradicated HCV, respectively. Conclusion: Compared to natives, HCV-infected migrants in care have different demographics, HCV genotypes, viral coinfections and comorbidities and similar disease severity, SVR and cofactors for disease progression after HCV eradication. A periodic clinical assessment after HCV eradication in Italians and migrants with cofactors for disease progression is warranted.

2021 - Domestic cooking methods affect the stability and bioaccessibility of dark purple eggplant (Solanum melongena) phenolic compounds [Articolo su rivista]
Martini, S.; Conte, A.; Cattivelli, A.; Tagliazucchi, D.
abstract

Eggplant is an important component of the Mediterranean Diet, which becomes edible after cooking. This study determined the fate of dark purple eggplant phenolic compounds after baking, boiling, frying, grilling and digestion. Thirty-seven phenolic compounds were identified and quantified in raw eggplant. Frying determined a 74% increase in total hydroxycinnamic acids whereas a decrease was observed after boiling (27%), grilling (51%), and baking (60%). After digestion, 45%, 33% and 22% of total phenolic compounds resulted bioaccessible in baked, grilled and fried dark purple eggplant. Fried eggplant displayed the highest amount of phenolic compounds (751.46 mg/100 g) after digestion. The cooking methods differently affected the release of individual phenolic compounds. Baking and grilling resulted in higher amount of bioaccessible caffeoylquinic acids whereas frying in di-caffeoylquinic acids and hydroxycinnamic acid-amides. A careful design of the cooking method may be pivotal to modulate the release of specific phenolic compounds.

2021 - Influence of cooking methods on onion phenolic compounds bioaccessibility [Articolo su rivista]
Cattivelli, A.; Conte, A.; Martini, S.; Tagliazucchi, D.
abstract

The impact of domestic cooking (baking, boiling, frying and grilling) and in vitro digestion on the stability and release of phenolic compounds from yellow-skinned (YSO) and red-skinned onions (RSO) have been evaluated. The mass spectrometry identification pointed out flavonols as the most representative phenolic class, led by quercetin-derivatives. RSO contained almost the double amount of phenolic compounds respect to YSO (50.12 and 27.42 mg/100 g, respectively). Baking, grilling and primarily frying resulted in an increased amount of total phenolic compounds, especially quercetin-derivatives, in both the onion varieties. Some treatments promoted the degradation of quercetin-3-O-hexoside-4′-O-hexoside, the main compound present in both the onion varieties, leading to the occurrence of quercetin-4′-O-hexoside and protocatechuic acid-4-O-hexoside. After in vitro digestion, the bioaccessibility index for total phenolic compounds ranged between 42.6% and 65.5% in grilled and baked YSO, respectively, and between 39.8% and 80.2% in boiled and baked RSO, respectively. Baking contributed to the highest amount of bioaccessible phenolic compounds for both the onion varieties after in vitro digestion. An in-depth design of the cooking process may be of paramount importance in modulating the gastro-intestinal release of onion phenolic compounds.

2021 - Peptidomics: new trends in food science [Articolo su rivista]
Martini, S.; Solieri, L.; Tagliazucchi, D.
abstract

In recent years, peptidomics is gaining ever-growing relevance in food science. The emerging field of food peptidomics is defined as the whole pool of peptides existing in food products or generated during food processing, storage or digestion. The recent advancement in high-resolution mass spectrometry techniques provides a more detailed picture, although not yet exhaustive, of the peptides present or derived from food. Food peptidomics techniques have been successfully applied in understanding food protein digestion, in the study of the microbial contribution to food protein hydrolysis, in the identification of food-derived bioactive peptides and peptide biomarkers. Ad-hoc efforts are still needed to improve the identification of short peptides and the analysis of the large data set generated.

2020 - Cultivable non-starter lactobacilli from ripened Parmigiano Reggiano cheeses with different salt content and their potential to release anti-hypertensive peptides [Articolo su rivista]
Tagliazucchi, Davide; Baldaccini, Andrea; Martini, Serena; Bianchi, Aldo; Pizzamiglio, Valentina; Solieri, Lisa
abstract

The impact of salt and fat intake on human health drives the consumer's attention towards dairy food with reduced salt and fat contents. How changes in salt and fat content modulate dairy LAB population and the associated proteolytic activities have been poorly studied. Here, non-starter LAB populations from 12 Parmigiano Reggiano (PR) cheeses (12-month ripened), clustered in low salt and fat content (LL-PR) and high salt and fat content (HH-PR) groups, were investigated and identified at specie-level with molecular assays. Lactobacillus rhamnosus was dominant in HH-PR samples, whereas Lactobacillus paracasei in LL-PR samples. (GTG)5 rep-PCR analysis discriminated 11 and 12 biotypes for L. rhamnosus and L. paracasei isolates, respectively. Screening for proteolytic activity identified L. rhamnosus strains more proteolytic than L. paracasei, and, within L. rhamnosus species, HH-PR strains were generally more proteolytic than LL-PR strains. Two L. rhamnosus representatives, namely strain 0503 from LL-PR and strain 2006 from HH-PR, were functionally characterized in cow milk fermentation assay. HH-PR strain 2006 overcame LL-PR strain 0503 in acidification performance, leading to a fermented milk with higher angiotensin I-converting enzyme inhibitory and antioxidant activities. L. rhamnosus 2006 was more prone to release VPP, while L. rhamnosus 0503 released higher amount of IPP. This study provides evidences that salt/fat content affects NSLAB cultivable fraction and the associated proteolytic ability resulting in a complex occurrence of bioactive peptides featuring health-promoting properties.

2020 - Effect of ripening and in vitro digestion on the evolution and fate of bioactive peptides in Parmigiano-Reggiano cheese [Articolo su rivista]
Martini, Serena; Conte, Angela; Tagliazucchi, Davide
abstract

The influence of ripening and in vitro digestion on the peptidomic profile of Parmigiano-Reggiano (PR) cheeses was investigated. Ripening and in vitro digestion thoroughly modified the peptidomic profile of the three cheeses. Twenty-six bioactive peptides were identified in undigested PR. Some peptides were degraded and others released during ripening. After digestion, 52 bioactive peptides were identified. Semi-quantitative data suggested that bioactive peptides released after digestion can be clustered in 5 groups according to the ripening time. VPP and IPP peptide levels in undigested samples were in the range of 4.52–11.34 and 0.66–4.24 mg kg−1, with the highest amounts found in 18-month ripened PR. YPFPGPI peptide was absent in undigested PRs but was released after digestion, especially in the 12-month-old sample (20.18 mg kg−1). The present study suggests possible differences in bioactive peptide levels after digestion as a function of the duration of ripening of PR cheese.

2020 - Functional, nutritional, antioxidant, sensory properties and comparative peptidomic profile of faba bean (Vicia faba, L.) seed protein hydrolysates and fortified apple juice [Articolo su rivista]
Samaei, S. P.; Ghorbani, M.; Tagliazucchi, D.; Martini, S.; Gotti, R.; Themelis, T.; Tesini, F.; Gianotti, A.; Gallina Toschi, T.; Babini, E.
abstract

Enzymatic hydrolysis of plant-derived proteins can improve their quality by offering opportunities for food applications. In this study, three proteolytic enzymes (pepsin, trypsin, Alcalase®) were used, alone or combined, to produce faba bean protein hydrolysates (PHs). Their functional, nutritional and antioxidant properties were evaluated, and the peptidomic profile was assessed by LC–MS/MS. Hydrolysis improved solubility of faba proteins at acidic and neutral pH, and their antioxidant properties. Peptidomic analysis identified 2031 peptides in the different PHs. Among them, 9 showed 100% homology with previously known antioxidant peptides and several others had antioxidant motifs in their sequences. Sensory data analysis showed that after addition of PHs to apple juice, no significant differences were perceived between control and some of the PHs. This study demonstrates that enzymatic hydrolysis enhances the functional and antioxidant properties of faba bean proteins. Specifically, hydrolysates can be used as functional food ingredients to produce fortified beverages.

2020 - Influence of linseed and antioxidant-rich diets in pig nutrition on lipidoxidation during cooking and in vitro digestion of pork [Articolo su rivista]
Martini, S.; Tagliazucchi, D.; Minelli, G.; Lo Fiego, D. P.
abstract

Enrichment of pig diets with polyunsaturated fatty acids (PUFA) is considered an emerging strategy to increasetheir intake in the human diet. However, PUFA are particularly vulnerable to oxidative reactions leading to thegeneration of toxic compounds. The aim of this study was to evaluate the effect of supplementation of pig dietswith extruded linseed (L), either or not in combination with synthetic antioxidants (E, tocopheryl-acetate andselenium) or natural extracts (P, grape-skin and oregano), and basal diet (C, without linseed) on the oxidativestability in raw, grilled andin vitrodigested pork. The diet supplementation with antioxidant-rich ingredientsresulted in the accumulation of specific metabolites in meat. Actually, 11 different phenolic- and 6 tocopherol-derived metabolites were identified by UHPLC/HR-MS. These metabolites were potentially correlated with thereduction in the oxidative phenomena occurring during meat cooking and digestion. Specifically, 16% and 35%reduction in the amounts of lipid hydroperoxides and TBA-RS were assessed after cooking of meat from P diet,respect to the L diet. Diet supplementations withα-tocopheryl acetate and selenium reduced the oxidativereactions only during meat cooking. A significant reduction was attended at the end ofin vitrodigestion, showingabout 24% and 34% hydroperoxides and TBA-RS concentration reductions, respectively, in P diet samples re-spect to the L ones. Thus, our study suggests that the appearance of phenolic metabolites in meat could beassociated to a reduction in the oxidative phenomena during meat cooking and digestion.

2020 - Mediterranean diet vegetable foods protect meat lipids from oxidation during in vitro gastro-intestinal digestion [Articolo su rivista]
Martini, S.; Conte, A.; Bottazzi, S.; Tagliazucchi, D.
abstract

Meat lipids oxidation during digestion gives rise to a post-prandial oxidative stress condition, which negatively affects human health. Mediterranean Diet vegetables contain high amount of phenolic compounds, which potentially may reduce the oxidative phenomena during digestion. In vitro co-digestion of turkey meat with a typical Mediterranean Diet salad containing tomato, onion, black olives, extra-virgin olive oil (EVOO) and basil, dose-dependently reduced lipid peroxidation. Onion and EVOO were more effective in limiting oxidation than the other foods, resulting in negligible concentrations of lipid hydroperoxides after digestion. Specific phenolic classes dominated the phenolic profile of the different foods, such as flavonols and anthocyanins in onion, phenolic acids in tomato and basil, and tyrosol-derivatives in black olives and EVOO. The correlation between lipid peroxidation inhibition, phenolic constituents and antioxidant properties was evaluated by principal component analysis (PCA). Flavonols and anthocyanin were the major contributors to the bioactive response of vegetable foods.

2020 - Microbial fermentation of industrial rice-starch byproduct as valuable source of peptide fractions with health-related activity [Articolo su rivista]
Babini, E.; Taneyo-Saa, D. L.; Tassoni, A.; Ferri, M.; Kraft, A.; Gran-Heedfeld, J.; Bretz, K.; Roda, A.; Michelini, E.; Calabretta, M. M.; Guillon, F.; Tagliazucchi, D.; Martini, S.; Nissen, L.; Gianotti, A.
abstract

The rice-starch processing industry produces large amounts of a protein-rich byproducts during the conversion of broken rice to powder and crystal starch. Given the poor protein solubility, this material is currently discarded or used as animal feed. To fully exploit rice’s nutritional properties and reduce this waste, a biotechnological approach was adopted, inducing fermentation with selected microorganisms capable of converting the substrate into peptide fractions with health-related bioactivity. Lactic acid bacteria were preferred to other microorganisms for their safety, efficient proteolytic system, and adaptability to different environments. Peptide fractions with different molecular weight ranges were recovered from the fermented substrate by means of cross-flow membrane filtration. The fractions displayed in vitro antioxidant, antihypertensive, and anti-tyrosinase activities as well as cell-based anti-inflammatory and anti-aging effects. In the future, the peptide fractions isolated from this rice byproduct could be directly exploited as health-promoting functional foods, dietary supplements, and pharmaceutical preparations. The suggested biotechnological process harnessing microbial bioconversion may represent a potential solution for many different protein-containing substrates currently treated as byproducts (or worse, waste) by the food industry.

2020 - Peptide profiling and biological activities of 12- month ripened parmigiano reggiano cheese [Articolo su rivista]
Tagliazucchi, D.; Solieri, L.; Baldaccini, A.; Martini, S.; Bianchi, A.; Pizzamiglio, V.
abstract

Proteolysis degree, biological activities, and water-soluble peptide patterns were evaluated in 12 month-ripened Parmigiano Reggiano (PR) cheeses collected in different dairy farms and showing different salt and fat content. Samples classified in high-salt and high-fat group (HH) generally showed lower proteolysis degree than samples having low-salt and low-fat content (LL). This positive correlation between salt/fat reduction and proteolysis was also confirmed by the analysis of biological activities, as the LL group showed higher average values of angiotensin-converting enzyme (ACE)-inhibitory and antioxidant activities. UHPLC/HR-MS allowed the identification of 805 unique peptides: LL and HH groups shared 59.3% of these peptides, while 20.9% and 19.9% were LL and HH specific, respectively. Frequency analysis of peptides identified a core of 183 peptides typical of 12-month ripened PR cheeses (corresponding to the 22.7% of total peptides), but no significant differences were detected in peptide patterns between LL and HH groups. Forty bioactive peptides, including 18 ACE-inhibitors and 12 anti-microbial peptides, were identified, of which 25 firstly found in PR cheese. Globally, this work contributed to unraveling the potentially healthy benefits of peptides fraction in PR cheese and provided prior evidence that PR with reduced at/salt content showed the highest antihypertensive and antioxidant activities.

2019 - Activity of extracts from three tropical plants towards fungi pathogenic to tomato (Solanum lycopersicum) [Articolo su rivista]
Mekam, Pascal Noel; Martini, Serena; Nguefack, Julienne; Tagliazucchi, Davide; Mangoumou, Ghislaine Ndonkeu; Stefani, Emilio
abstract

Antifungal properties were assessed of water and ethanol extracts from the pan-tropical plants Oxalis barrelieri L., Stachytarpheta cayennensis L., and Euphorbia hirta L. against Fusarium oxysporum f. sp. vasinfectum, Alternaria solani Sorauer, and Rhizoctonia solani Kuhn. The plant extracts inhibited fungal growth in vitro at 1.25-20 mg mL-1, and the degrees of inhibition increased in a dose-dependent manner. Ethanol extracts from the plants inhibited fungal growth by 80-100%, while water extracts showed less antifungal activity, with maximum growth inhibition of 62%. Growth inhibition from ethanol extracts was two- to three-fold greater than for water extracts at equivalent concentrations. Antifungal activity of the extracts varied with their content and composition of phenolics, flavonoids, tannins, and alkaloids. In greenhouse experiments, spraying tomato plants (Solanum lycopersicum L.) with ethanol extract from E. hirta at 2.5 mg mL-1 did not cause phytotoxicity, and increased plant size, when compared to untreated plants. Spraying E. hirta ethanol extract on tomato plants infected by R. solani reduced disease severity up to 80%, when compared to non-sprayed plants. These results demonstrate potential of leaf extracts from E. hirta, O. barrelieri, and S. cayennensis as biofungicides for the control of R. solani, A. solani, and F. oxysporum, which are among the most important causal agents of tomato diseases.

2019 - Antiproliferative Activity and Cell Metabolism of Hydroxycinnamic Acids in Human Colon Adenocarcinoma Cell Lines [Articolo su rivista]
Martini, Serena; Conte, Angela; Tagliazucchi, Davide
abstract

In this study, we investigated the antiproliferative activity and the stability and metabolic fate of the main dietary hydroxycinnamates, using two colonic adenocarcinoma cell models (Caco-2 and SW480). Dihydrocaffeic and dihydroferulic acids were the most effective against cell proliferation in both cell lines with IC50 values of 71.7 ± 1.1 and 83.1 ± 1.1 μmol/L, respectively (P < 0.05) in Caco-2. At 200 μmol/L, caffeic and ferulic acids inhibited SW480 proliferation by 40.8 ± 1.6 and 59.9 ± 1.3%, respectively. Hydroxycinnamic acids with a catechol-type structure were degraded in Caco-2 cell medium, resulting in the production of H2O2. Intracellular Caco-2 UDP-glucuronosyltransferases and catechol-O-methyltransferases were able to form glucuronide and methyl conjugates. However, only the sulfate conjugates were detected after incubation with SW480. In addition, simple hydroxycinnamates were released from quinic and aspartic conjugates. The remarkable effect of dihydrocaffeic and dihydroferulic acids against cell proliferation is of paramount importance, since these compounds are the main metabolites detectable at the colonic level.

2019 - Bioactivity and cell metabolism of in vitro digested sweet cherry (Prunus avium) phenolic compounds [Articolo su rivista]
Martini, Serena; Conte, Angela; Tagliazucchi, Davide
abstract

In this study, the bioaccessibility of phenolic compounds after in vitro gastrointestinal digestion of two cherry cultivars was assessed. The phenolic profile was modified during in vitro digestion, with a considerable decrease of total and individual phenolic compounds. Hydroxycinnamic acids and especially coumaroylquinic acids showed the highest bioaccessibility. Isomerisation of caffeoylquinic and coumaroylquinic acids was observed after in vitro digestion. Modification of the phenolic profile after digestion resulted in an increased or decreased scavenging activity depending on the assay. In vitro digested phenolic-rich fractions also showed antiproliferative activity against SW480 but no effect against Caco-2 cell lines. Both Caco-2 and SW480 cell lines were able to metabolise cherry phenolic compounds with remarkable differences. An accumulation of glycosylated flavonols was observed in SW480 medium. In conclusion, phenolic compounds from cherries and especially hydroxycinnamic acids were efficiently released and remained bioaccessible after in vitro digestion, resulting in antioxidant and antiproliferative activities.

2019 - Bioprospecting for Bioactive Peptide Production by Lactic Acid Bacteria Isolated from Fermented Dairy Food [Articolo su rivista]
Tagliazucchi, Davide; Martini, Serena; Solieri, Lisa
abstract

With rapidly ageing populations, the world is experiencing unsustainable healthcare from chronic diseases such as metabolic, cardiovascular, neurodegenerative, and cancer disorders. Healthy diet and lifestyle might contribute to prevent these diseases and potentially enhance health outcomes in patients during and after therapy. Fermented dairy foods (FDFs) found their origin concurrently with human civilization for increasing milk shelf-life and enhancing sensorial attributes. Although the probiotic concept has been developed more recently, FDFs, such as milks and yoghurt, have been unconsciously associated with health-promoting effects since ancient times. These health benefits rely not only on the occurrence of fermentation-associated live microbes (mainly lactic acid bacteria; LAB), but also on the pro-health molecules (PHMs) mostly derived from microbial conversion of food compounds. Therefore, there is a renaissance of interest toward traditional fermented food as a reservoir of novel microbes producing PHMs, and “hyperfoods” can be tailored to deliver these healthy molecules to humans. In FDFs, the main PHMs are bioactive peptides (BPs) released from milk proteins by microbial proteolysis. BPs display a pattern of biofunctions such as anti-hypertensive, antioxidant, immuno-modulatory, and anti-microbial activities. Here, we summarized the BPs most frequently encountered in dairy food and their biological activities; we reviewed the main studies exploring the potential of dairy microbiota to release BPs; and delineated the main effectors of the proteolytic LAB systems responsible for BPs release

2019 - Comparative peptidomic profile and bioactivities of cooked beef, pork, chicken and turkey meat after in vitro gastro-intestinal digestion [Articolo su rivista]
Martini, Serena; Conte, Angela; Tagliazucchi, Davide
abstract

This study was designed to investigate the potential contribution of bioactive peptides to the biological activities related to the consumption of pork, beef, chicken and turkey meat following in vitro gastro-intestinal digestion. After extraction of the peptidic fractions from digested samples, the bioactivities were evaluated by in vitro antioxidant activity as well as angiotensin-converting enzyme (ACE) and dipeptidyl peptidase-IV (DPP-IV) inhibition assays. Pork and turkey meat appeared to be the best sources of antioxidant peptides. Pork was found to be the best source of DPP-IV-inhibitory peptides whereas chicken meat supplied peptides with the highest ACE-inhibitory activity. The comprehensive analysis of the peptidomic profile of digested samples was performed by nano-LC-ESI-QTOF MS/MS analysis. A total of 217, 214, 257 and 248 peptides were identified in digested pork, beef, chicken and turkey meat, respectively. Chicken and turkey meat showed the highest similarity in peptide sequences with 202 common peptides. Sixty-two peptides matched with sequences with previously demonstrated biological activity. In particular, 35 peptides showed ACE-inhibitory activity and 23 DPP-IV inhibitory activity. Twenty-two bioactive peptides were commonly released from the different types of meat. The relative amount of identified bioactive peptides were positively correlated to the biological activities of the different digested meats. Biological significance: The present study describes for the first time a comprehensive peptide profile of four types of meat after in vitro gastro-intestinal digestion. The peptide inventory was used to identify 62 bioactive peptides with ACE- and DPPIV-inhibitory and antioxidant activities. The bioactivity analysis revealed interesting and significant differences between the studied meats. The originality of this work lay in the description of intrinsic differences in physiological functions after the ingestion of meat proteins from different species. In a context in which the current research scene relates meat consumption to the onset of chronic pathologies, this peptide profiling and bioactivity analysis shed light on the possible health benefits of peptides released from meat proteins. In fact, this paper represents a sort of detailed peptide list that may help to predict which peptides could be generated after meat intake and detectable at gastro-intestinal level. It also provides a thorough investigation of novel biological activities associated to meat protein hydrolysates, giving a new positive aspect to meat consumption.

2019 - Phenolic compounds profile of water and ethanol extracts of Euphorbia hirta L. leaves showing antioxidant and antifungal properties [Articolo su rivista]
Mekam, Pascal Noel; Martini, Serena; Nguefack, Julienne; Tagliazucchi, Davide; Stefani, Emilio
abstract

The bioactive chemical constituents of water and ethanol extracts of Euphorbia hirta L. leaves have been identified and quantified using an un-targeted mass spectrometric approach. The study allowed the tentative identification of 123 individual phenolic compounds and 18 non-phenolic phytochemicals, most of them described in Euphorbia hirta L. leaves for the first time. Gallotannins, hydroxybenzoic and hydroxycinnamic acids were the most abundant phenolic classes in Euphorbia hirta L. leaves, representing together the 71.5% (26.3%, 25.2% and 20%, respectively) of the total amount of identified phenolics. The main phenolic compounds detected were tri-O-galloyl-glucose isomers, feruloyl-coniferin, tetra-O-galloyl-glucose isomers, di-O-galloyl-glucose isomers, ethyl-gallic acid, protocatechuic acid-O-pentoside-O-hexoside, 5-O-caffeoyl-quinic acid trans isomer and digalloyl-quinic acid. Feruloyl-coniferin was found to be approximately six times more concentrated in the ethanol extract with respect to the water extract. The ethanol extract exhibited higher ABTS (1338.3 ± 85.3 and 802.3 ± 91.0 μmol ascorbic acid equivalent/gram of dry extract, respectively) and superoxide anion (2014.6 ± 78.6 and 1528.0 ± 111.7 μmol ascorbic acid equivalent/gram of dry extract, respectively) scavenging abilities than the water extract. Additionally, the ethanol extract also showed a remarkable anti-fungal effect against Fusarium oxysporum f. sp. vasinfectum, Alternaria solani and Rhizoctonia solani. This study provides new information about Euphorbia hirta L., offering reasons to promote this plant species as rich source of phenolics and an excellent source of antifungal molecules that might have a prospective use in controlling fungal diseases of vegetable crops.

2019 - Protocatechuic and 3,4-Dihydroxyphenylacetic Acids Inhibit Protein Glycation by Binding Lysine through a Metal-Catalyzed Oxidative Mechanism [Articolo su rivista]
Tagliazucchi, Davide; Martini, Serena; Conte, Angela
abstract

The mechanism of inhibition of advanced glycation end product (AGE) formation by protocatechuic acid and 3,4-dihydroxyphenylacetic acid (DHPA) has been studied using a widespread applied in vitro model system composed of bovine serum albumin (BSA) and supraphysiological glucose concentrations. Protocatechuic acid and DHPA inhibited the formation of Amadori compounds, fluorescent AGEs (IC50 = 62.1 ± 1.4 and 155.4 ± 1.1 μmol/L, respectively), and Nε-(carboxymethyl)lysine (IC50 = 535.3 ± 1.1 and 751.2 ± 1.0 μmol/L, respectively). BSA was pretreated with the two phenolic acids, and the formation of BSA–phenolic acid adducts was estimated by nanoflow liquid chromatography–electrospray ionization–quadrupole time-of-flight mass spectrometry. Results showed that the tested phenolic acids bound key sites of glycation in BSA through a metal-catalyzed oxidative mechanism. The antiglycative activity mechanism involved the formation of BSA–phenolic acid adducts, and it is unlikely that this occurs in vivo. These results raise the problem to design in vitro models closer to physiological conditions to reach biologically sound conclusions.

2018 - Bioaccessibility, bioactivity and cell metabolism of dark chocolate phenolic compounds after in vitro gastro-intestinal digestion [Articolo su rivista]
Martini, Serena; Conte, Angela; Tagliazucchi, Davide
abstract

The bioaccessibility of phenolic compounds after in vitro gastro-intestinal digestion of dark chocolate, dark chocolate enriched with Sakura green tea and dark chocolate enriched with turmeric powder was studied. The phenolic profile, assessed by accurate mass spectrometry analysis, was modified during in vitro gastro-intestinal digestion, with a considerable decrease of total and individual phenolic compounds. Phenolic acids showed the highest bioaccessibility with hydroxycinnamic acids displaying higher bioaccessibility (from 41.2% to 45.1%) respect to hydroxybenzoic acids (from 28.1% to 43.5%). Isomerisation of caffeoyl-quinic acids and galloyl-quinic acids as well as dimerization of (epi)gallocatechin were also observed after in vitro gastro-intestinal digestion. Antioxidant activity increased after the gastric step and rose further at the end of the digestion. Furthermore, in vitro digested phenolic-rich fractions showed anti-proliferative activity against two models of human colon adenocarcinoma cell lines. Cell metabolism of digested phenolic compounds resulted in the accumulation of coumaric and ferulic acids in the cell media.

2018 - Biological activities and peptidomic profile of in vitro-digested cow, camel, goat and sheep milk [Articolo su rivista]
Tagliazucchi, Davide; Martini, Serena; Shamsia, Sherif; MOHAMED IBRAHIM HELAL, Ahmed; Conte, Angela
abstract

In vitro digestibility, selected biological activities and digested products of proteins from skimmed cow, camel, goat and sheep milk were compared. The harmonised in vitro INFOGEST digestion model and mass spectrometry were combined to identify peptides. Goat milk had the highest digestibility, sheep milk the highest angiotensin-converting enzyme (ACE)-inhibitory activity after digestion and cow milk the highest dipeptidyl peptidase-IV (DPP-IV)-inhibitory activity. After in vitro digestion of the milk samples, 522 peptides were identified. Goat and sheep milk showed the highest peptide sequence similarity with 151 common peptides. Thirteen, forty-three and twenty peptides with previously demonstrated antioxidant, ACE-inhibitory and DPP-IV-inhibitory activities, respectively, were found. Nineteen bioactive peptides in common were released from the different milk types. Despite limitations related to analysis of one milk sample for each species, possible differences in physiological functions after ingestion of milk from different species are suggested; this requires confirmation by in vivo testing.

2018 - Comprehensive evaluation of phenolic profile in dark chocolate and dark chocolate enriched with Sakura green tea leaves or turmeric powder [Articolo su rivista]
Martini, Serena; Conte, Angela; Tagliazucchi, Davide
abstract

Recently, a huge number of studies have confirmed the important role of chocolate polyphenols in human health, underlining its beneficial effects especially in the treatment of cardiovascular diseases. However, a thorough evaluation of chocolate phenolic profile is still lacking. This study aimed at a comprehensive characterization of dark chocolate phenolic profile, using non-targeted mass spectrometry identification. This approach allowed a tentative identification of 158 individual phenolic compounds: 67 were newly detected in dark chocolate, among these 38 were observed for the first time in chocolate as well as in cocoa beans or products. Ellagitannins, which have never been reported in cocoa or chocolate, represented about the 10% of the phenolic profile of dark chocolate. The enrichment of dark chocolate with Sakura green tea leaves or turmeric powder influenced and modified the phenolic profile, resulting in a phenolic concentration increase. In this way, this functional chocolate might maximize the beneficial effect of chocolate consumption, combining the positive health effects of chocolate, turmeric and green tea and, at the same time, reducing the amount of sugars and calories introduced with chocolate.

2018 - The paradoxical effect of extra-virgin olive oil on oxidative phenomena during in vitro co-digestion with meat [Articolo su rivista]
Martini, Serena; Cavalchi, Martina; Conte, Angela; Tagliazucchi, Davide
abstract

Extra-virgin olive oil is an integral part of the Mediterranean diet and its consumption has been associated with a reduction risk of chronic diseases. Here we tested the potential of extra-virgin olive oil to limit the oxidative phenomena during in vitro gastro-intestinal co-digestion with turkey breast meat. The extra-virgin olive oil was particularly rich in oleuropein aglycone isomers, which represented the 66.8% of total phenolic determined with MS/MS experiments. Meals supplemented with extra-virgin olive oil equivocally affected lipid peroxidation. At low concentration (2.5% respect to meat), a significant inhibition of lipid oxidation was observed, whereas lipid peroxidation was greatly enhanced when the amount of extra-virgin olive oil was increased in the gastro-intestinal system. The inhibitory effect observed at 2.5% extra-virgin olive oil was due to the antioxidant properties of extra-virgin olive oil phenolic compounds. At high concentration, extra-virgin olive oil phenolic compounds (especially hydroxytyrosol-derivative) behaved as pro-oxidants increasing the generation of lipid hydroperoxides from meat. At the same time, the presence in the digestive system of catalysers from meat induced the peroxidation of extra-virgin olive oil fatty acids, which was further intensified by the pro-oxidant activity of extra-virgin olive oil phenolic compounds. Our study underlined the importance of the timing and amount of consumption of extra-virgin olive oil as well as its phenolic composition in limiting the peroxidative phenomena on meat lipids during digestion.

2017 - ANTIFUNGAL ACTIVITY OF AQUEOUS AND ETHANOL EXTRACTS OF TROPICAL PLANTS AGAINST TOMATO FUNGAL PATHOGENES. [Abstract in Atti di Convegno]
Mekam, Pascal Noel; Martini, Serena; Tagliazucchi, Davide; Nguefack, Julienne; Stefani, Emilio
abstract

Several plant extracts may have a strong antifungal activity that can be exploited in the management of fungal diseases as an alternative to synthetic fungicides, the abuse of which may have consequences on the environment and health. Ethanol extracts (EE) and water extracts (WE) of three tropical plants, Stachytarpheta cayennensis (Verbenaceae), Oxalis barrelieri (Oxalidaceae) and Euphorbia hirta (Euphorbiaceae) were obtained and screened for their antifungal activity against three major phytopathogenic fungi of tomato: Fusarium sp., Alternaria sp. and Colletotrichum sp. The phytopathogenic fungi used across the experiments were isolated in Cameroon on severely affected tomatoes and their molecular identification and characterization is ongoing. The antifungal activity was checked in vitro on different media supplemented with different concentrations of the extracts. O. barrelieri EE, at the concentration of 25 mg/ml, inhibited the mycelium growth of Fusarium sp. by 26.8% and remarkably modified its morphology. WE and EE of the same O. barrelieri inhibited the mycelium growth of Colletotrichum sp. by 43.7% and 50.3% respectively, but showed no inhibition on Alternaria sp. The phytochemical analysis of these plant extracts revealed that EE of S. cayennensis was the richest in polyphenols and flavonoids. EE from O. barrelieri was particularly rich in alkaloids. The inhibitory effects on the phytopathogenic fungi were possibly related to the amount of polyphenols and alkaloids obtained through the extraction. Field experiments are being conducted on tomato to confirm the action of such extracts in vivo. These findings may contribute to develop new biofungicides to protect tomato from some fungal pathogens.

2017 - Chocolate melanoidins as a carrier for the delivery of phenolic compounds in the gut [Poster]
Martini, S.; Conte, A.; Tagliazucchi, D.
abstract

Emerging evidence from in vitro and in vivo studies suggested that the gastro-intestinal tract may be the key site for the biological action of food melanoidins [1]. In this work, we tested high molecular weight melanoidins (HMWM) extracted from dark chocolate for their ability to release low molecular weight polyphenols following in vitro digestion. Water soluble HMWM were extracted by ultrafiltration (10 kDa) from three different types of dark chocolate (dark 70% cocoa (DC), dark 70% cocoa and 8% turmeric (TDC), dark 62% cocoa and 2% Sakura green tea (GTDC)). HMWM have been chemically characterized for their content in total polyphenols, protein, and polysaccharides [2]. HMWM were in vitro digested and separated from low molecular weight fractions (LMWF) by ultrafiltration. Digested HMWM were chemically characterized and LMWF were characterized for their total phenolic content. Individual phenolic compounds in the LMWF were identified and quantified by HPLC-ESI-QTOF-MS/MS. Results showed that melanoidins are carbohydrate-based structures with the phenolic content ranging from 8.62 ± 1.0 in TDC to 12.79 ± 0.4 g/100 g HMWM in GTDC. The in vitro digestion caused a general decrease in the polysaccharide, protein and phenolic content. Digestion resulted in the release of phenolic compounds from HMWM to LMWF. The highest amount was recovered in the LMWF of GTDC (436.2 ± 87.6 mg/L), whereas the lowest amount in the LMWF of TDC (242.1 ± 17.1 mg/L). The most representative classes of polyphenols released from HMWM were stilbenes (resveratrol and derivatives) and phenolic acids (hydroxybenzoic, benzoic, quinic, cinnamic, coumaric and ferulic acids). As expected, food matrices influenced the chemical composition of HMWM. Specific phenolic compounds were detected in the LMWF from GTDC (quercetin-3-rutinoside) and TDC (curcumin, cathecol and protocatechuic acid).

2017 - Identification and in vitro bioaccessibility of dark chocolate phenolic compounds [Poster]
Martini, S.; Conte, A.; Tagliazucchi, D.
abstract

Polyphenols have become an intense focus of research interest because of their perceived health-beneficial effects such as anti-atherogenic, anti-inflammatory and anti-tumoral effects. Cocoa (Theobroma cacao) is a major economically important international crop and has been associated with several nutritional benefits [1]. The aim of the present study was to identify and quantify phenolic compounds from three different types of dark chocolate using HPLC-ESI-QTOF-MS/MS. Phenolic compounds were also identified and quantified after in vitro gastro-intestinal digestion. Three different types of dark chocolate (dark 70% cocoa (DC), dark 70% cocoa and 8% turmeric (TDC), dark 62% cocoa and 2% Sakura green tea (GTDC)) were in vitro digested with a simulated gastro-intestinal procedure [2]. Polyphenols were extracted from un-digested samples with methanol-water-formic acid solution (70:28:2 v/v) followed by a second extraction step with acetone. Total polyphenols were determined by Folin-Ciocalteau assay whereas antioxidant activity by FRAP methods. Individual phenolic compounds were identified and quantified using mass spectrometry. Total polyphenol concentration in the un-digested samples varied from 2.6 ± 0.1 in DC to 3.4 ± 0.2 g/100g of chocolate in GTDC. The antioxidant activity ranged between 6.3 ± 0.1 to 15.4 ± 0.7 mmol trolox/100g in DC and GTDC, respectively. As expected, monomeric and oligomeric flavan-3-ols were the most representative compounds in all the three samples, followed by resveratrol and derivative. In addition, some phenolic acids (caffeoylquinic, protocatechuic, vanillic acids) and ellagic acid derivative (ellagic acid, ellagic acid hexoside) were also identified. The addition of green tea and turmeric resulted in the presence of gallate flavan-3-ols (such as epigallocatechin-3-gallate, epigallocatechin and epicatechin gallate) and curcuminoids, respectively. In vitro digestion resulted in a decrease in total polyphenols and in the individual phenolic profile modification.

2017 - LC-ESI-QTOF-MS identification of novel antioxidant peptides obtained by enzymatic and microbial hydrolysis of vegetable proteins [Articolo su rivista]
Babini, Elena; Tagliazucchi, Davide; Martini, Serena; Più, Lucilla Dei; Gianotti, Andrea
abstract

Bioactive antioxidant peptides are more and more attracting the attention of food manufacturers for their potential to transform food in functional food, able to prevent a variety of chronic diseases associated with oxidative stress. In the present study proteins extracted from different vegetable sources (KAMUT khorasan wheat, emmer, lupine and pea) were hydrolyzed with commercial enzymes and Lactobacillus spp. strains. Hydrolysates were separated by size exclusion chromatography and purified fractions were analyzed for their antioxidant activity. Peptides from the fractions with the highest activity were identified by nanoLC-ESI-QTOF-MS and thirteen peptides were selected for synthesis on the basis of their sequence. Four peptides (VLPPQQQY, TVTSLDLPVLRW, VTSLDLPVLRW, FVPY) were found able to scavenge superoxide anion and hydroxyl radicals, organic nitro-radicals (ABTS, DPPH) and to inhibit lipid peroxidation. The impact of this work is targeted to add hydrolysed vegetable proteins to reformulated functional food or to produce health-promoting ingredients and nutraceuticals.

2017 - PEPTIDOMIC PROFILE OF IN VITRO DIGESTED BOVINE, CAMEL, GOAT AND SHEEP MILK [Abstract in Atti di Convegno]
Tagliazucchi, Davide; Martini, Serena; Sherif, Shamsia; Ahmed, Helal; Conte, Angela
abstract

Milk and dairy products have long traditions in human nutrition since they are a rich source of nutrients such lipids, proteins, amino acids, vitamins and minerals. Nowadays, milk may also be considered as a source of health-promoting compounds. Bioactive peptides deriving from milk proteins may play an important role in the prevention and treatment of metabolic syndrome and its complications [1]. This study aimed to characterize the peptidomic profile of in vitro digested skimmed bovine, camel, goat and sheep milk. Milk samples were in vitro digested following the harmonized INFOGEST protocol [2]. The peptide fractions were extracted from fully digested milk by ultrafiltration (cut-off 3 kDa) and the permeate were characterized by nanoflow-LC-ESI-QTOF MS/MS analysis. Digestibility and biological activities of the peptide fractions were also determined at the end of the digestion. The results showed a faster and more efficient gastric and duodenal degradation of goat milk proteins than camel, bovine and sheep milk. More than 100 peptides were identified in the permeate of digested milk, most of them arising from beta-casein. Goat, sheep and bovine milk showed the highest similarity in peptide sequences than camel milk. Peptides with previously demonstrated biological activities were found in the permeate of all the milk samples after in vitro digestion. Biological activities analysis showed that bovine milk peptides were the most effective in scavenging hydroxyl radical and in the inhibition lipid peroxidation. Bovine milk was also the best source of DPPIV-inhibitory peptides (IC50=6.87 mg of peptides/mL) whereas sheep milk was the best source of ACE-inhibitory peptides (IC50=625.4 μg of peptides/mL). The quantitative diversity in the identified bioactive peptides explained the observed differences in the biological activities.

2017 - Phenolic compounds profile and antioxidant properties of six sweet cherry (Prunus avium) cultivars [Articolo su rivista]
Martini, Serena; Conte, Angela; Tagliazucchi, Davide
abstract

Sweet cherry (Prunus avium) fruits are a nutritionally important food rich in dietary phenolic compounds. The aim of this study was to investigate the phenolic pro file and chemometric discrimination of fruits from six cherry cultivars using a quantitative metabolomics approach, which combine non-targeted mass spectrometry and chemometric analysis. The assessment of the phenolic fingerprint of cherries allowed the tentative identi fication of 86 compounds. A total of 40 chlorogenic acids were identi fied in cherry fruit, which pointed out hydroxycinnamic acid derivatives as the main class of phenolics by number of compounds. Among the compounds detected, 40 have been reported for the first time in sweet cherry fruit. Hydroxycinnamic acids are also the quantitatively most represented class of phenolic compounds in the cherry cultivars with the exception of Lapins and Durone della Marca where the most representative class of phenolic compounds were anthocyanins and flavan-3-ols, respectively. This non-targeted approach allowed the tentative identi fication of the cultivar-compound relationships of these six cherry cultivars. Both anthocyanins and colorless phenolic compounds profile appeared to be cultivar-dependent. In detail, anthocyanins and flavonols patterns have the potential to be used for the determination of a varietal assignment of cherries

2016 - Bioaccesssibility and bioactivity of polyphenols extracted from six cherry cultivars [Poster]
Martini, S.; Tagliazucchi, D; Conte, A
abstract

Introduction There is a possible association between consumption of fruit and vegetables and reduced risk of cancer, particularly cancer of the digestive tract. This anti-cancer activity has been attributed in part to polyphenols present in foods [1]. Cherries in particular are a rich source of polyphenols, especially anthocyanins and hydroxycinnamic acids [2]. Method Cherries from six different cultivars (Durone della Marca, Celeste, Durone Nero I, Bigarreau, Lapins and Moretta) were in vitro digested with a procedure that mimicked the physiochemical conditions of the gastro-intestinal tract [3]. After digestion, polyphenol-rich extracts were obtained by preparative chromatography on C18 column. Polyphenols were also extracted from un-digested cherries with methanol-water-formic acid solution (70:28:2 v/v) and chromatographed on C18 column. Total polyphenols and anthocyanins concentration were determined by Folin-Ciocalteau assay and pH differential method, respectively [3]. Polyphenols were identified using liquid chromatography mass spectrometry. The digested and un-digested preparations were assessed for their cytotoxic and anti-proliferative activities using two human colon adenocarcinoma cell lines (Caco-2 and SW480) and IC50 values of anti-proliferative activity were determined. Results / Discussion / Conclusion The total polyphenol concentration in the un-digested samples varied from 70.2mg/100g of cherry in the cultivar Durone della Marca to 280.6mg/100g of cherries in the cultivar Durone Nero I. The total anthocyanin content varied from 0.12mg/100g of cherry in the cultivar Durone della Marca to about 17mg/100g of cherry in the two darker cultivars (Lapins and Moretta). In vitro digestion resulted in a decrease in both total polyphenols and anthocyanins. Total polyphenols decrease ranged from 56 to 80%, whereas total anthocyanin decrease was more than 90% in all the tested cultivars. Mass spectrometry showed that the gastro-intestinal digestion decreased the content of hydroxycinnamic acids and anthocyanins. Other classes of polyphenols are more stable to digestion. Breakdown products of polyphenols are also present. Digested and un-digested polyphenol-rich extracts showed no cytotoxic activity. Un-digested polyphenol-rich extract from the cultivar Bigarreau showed the highest anti-proliferative effect (IC50 of 7.1 ± 1.1 µg polyphenols/mL) on the SW480 cells. The in vitro digestion increased the anti-proliferative activity of cherry extracts. Polyphenol-rich extracts from digested Durone della Marca and Celeste were the most active with IC50 values of 1.7 ± 0.8 and 3.5 ± 1.2 µg polyphenols/mL, respectively. All of the tested extracts showed no anti-proliferative activity on Caco-2 cells. Our results showed that cherry polyphenols were effective anti-proliferative agents on the more undifferentiated colon adenocarcinoma cells SW480 and that their digestion increased the effect. The increase may be due to the formation of polyphenol metabolites. A great difference was observed between the cultivars used. Further research is required to identify the compounds responsible for the observed anti-proliferative effect.

2016 - Colon-available dietary melanoidins exhibit cytotoxic activity on human colon adenocarcinoma cell lines [Poster]
Tagliazucchi, D.; Martini, S.; Conte, A
abstract

Introduction Emerging evidence from in vitro and in vivo studies suggested that the gastro-intestinal tract might be the key site for the biological action of food melanoidins [1]. Recently, Vitaglione et al. [2] reviewed the possible mechanisms by which coffee melanoidins may influence the risk of colorectal cancer development. In this work, we tested the water-soluble high molecular weight melanoidins (HMWM) obtained from different food sources for their cytotoxic activity against colon cancer cell lines. Method HMWM were obtained by ultrafiltration (cut-off 10 kDa) from instant coffee, cocoa brew, dark beer and instant barley coffee beverages [2]. HMWM have been chemically characterized for their content in total polyphenols, protein, and polysaccharides as well as their spectroscopic properties. The value of Kmix 420nm (defined as the absorption at 420nm of a solution of HMWM at a concentration of 1 g/L) provides information on the relative amount of melanoidins (Kmix 420nm) respect to other compounds found in the HMWM of beverages. Cytotoxic activity against two colon cancer cell lines (Caco-2 and SW480) was assayed by MTT assay. HMWM obtained from the above beverages submitted to in vitro gastro-intestinal digestion [3], were subjected to the same chemical analysis and assayed for their cytotoxic activity. Results / Discussion / Conclusion Barley coffee had the highest HMWM content (83.3g/100g of dry matter) followed by coffee (34.4g/100g of dry matter), cocoa (28.1g/100g of dry matter) and dark beer (20.0g/100g of dry matter). The HMWM of coffee had the highest Kmix 420nm value, suggesting that this fraction is relatively rich in melanoidins. On the contrary, despite the highest content in HMWM, barley coffee was the sample with the lowest melanoidins content. Protein content of HMWM ranged from 6.1% (dark beer) to 10.6% (coffee) whereas polysaccharides content ranged from 42% (dark beer) to 61% (cocoa). Cocoa and coffee HMWM had the highest phenolic content (6 and 12%, respectively). The in vitro digestion caused a general decrease in the polysaccharide and protein content of all the analysed HMWM because of the action of amylolytic and proteolytic enzymes. The analysis of the content of polyphenols showed a decrease in these components as a result of the digestion. On the contrary, the in vitro digestion caused an increase in the Kmix 420nm of all the HMWM investigated suggesting that this process resulted in an enrichment in melanoidins. The effect of HMWM on the human colon adenocarcinoma Caco-2 and SW480 cell cultures was investigated. Un-digested HMWM had no significant cytotoxic effect against both the cell lines. However, the in vitro digestion greatly increased the cytotoxic activity of HMWM against both the cell lines. Coffee HMWM were the most active against Caco-2 cells whereas cocoa HMWM were found to be most active against SW480 cells. Considering that the colon accumulates its content over at least 24 h in a maximum volume of 2 l, it is possible to estimate the concentrations of HMWM reaching daily the colon with diet according to the different food matrices [1]. Data showed that, the consumption of coffee, cocoa and barley coffee beverages may result in intestinal concentrations of HMWM, which are cytotoxic against Caco-2 and SW480 cell lines.

2016 - Release of the Antihypertensive Tripeptides Valine-Proline-Proline and Isoleucine-Proline-Proline from Bovine Milk Caseins during in Vitro Gastrointestinal Digestion [Articolo su rivista]
Rutella, GIUSEPPINA SEFORA; Solieri, Lisa; Martini, Serena; Tagliazucchi, Davide
abstract

The aim of this study was to identify and quantify the release of antihypertensive tripeptides valine-proline-proline (VPP) and isoleucine-proline-proline (IPP) during in vitro oro-gastro-intestinal (OGI) digestion of bovine skimmed milk. The experimental approach combined the recently developed harmonized static in vitro digestion (IVD) model and targeted mass spectrometry to monitor peptide generation. We first demonstrated that VPP and IPP are released from bovine milk proteins during in vitro OGI digestion at final concentrations of 354.3 ± 29.8 and 973.8 ± 155.7 μg/L, respectively. In silico analysis of cleavage sites and mass spectrometry revealed that tetrapeptides VPPF, IPPL, and IPPK are precursors of VPP and IPP. The release of other ACE-inhibitory peptides, such as FVAP, VAP, AW, and VY, was demonstrated, and their fate and the time course were investigated. This research underlines the suitability of an IVD system to study the release of short bioactive peptides during OGI transit.

2015 - Identification of ACE-inhibitory peptides from Phaseolus vulgaris after in vitro gastrointestinal digestion [Articolo su rivista]
Tagliazucchi, Davide; Martini, Serena; Bellesia, Andrea; Conte, Angela
abstract

The objective of this study was to identify the angiotensin I-converting enzyme (ACE)-inhibitory peptides released from thermally treated Phaseolus vulgaris (pinto) whole beans after in vitro gastrointestinal digestion. The degree of hydrolysis increased during digestion reaching a value of 50% at the end of the pancreatic digestion. The<3 kDa fraction of the postpancreatic sample showed high ACE-inhibitory activity (IC50=105.6±2.1 mug of peptides/mL). Peptides responsible for the ACE-inhibitory activity were isolated by reverse-phase high-performance liquid chromatography (HPLC). Three fractions, showing the highest inhibitory activity, were selected for tandem mass spectrometry (MS/MS) experiments. Eleven of the identified sequences have previously been described as ACE-inhibitors. Most of the identified bioactive peptides have a hydrophobic amino acid, (iso)leucine or phenylalanine, or proline at the C-terminal position, which is crucial for their ACE-inhibitory activity. The sequence of some peptides allowed us to anticipate the presence of ACE-inhibitory activity.