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ALICE CATTIVELLI

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2023 - Effect of Ripening and In Vitro Digestion on Bioactive Peptides Profile in Ras Cheese and Their Biological Activities [Articolo su rivista]
Helal, A.; Cattivelli, A.; Conte, A.; Tagliazucchi, D.
abstract

The effect of ripening and in vitro digestion on the biological activities, peptide profiles and release of bioactive peptides in Ras cheese has been investigated. Ras cheese ripening largely influenced the extent of protein hydrolysis. The advancement in ripening resulted in an increase in total peptides (from 0.97 to 2.46 mmol leucine/g in samples at 30 and 180 days of ripening, respectively) and bioactive peptides concentration, especially angiotensin-converting enzyme (ACE)-inhibitory, dipeptidyl-peptidase-IV-(DPP-IV)-inhibitory and antioxidant peptides. In vitro gastro-intestinal digestion further promoted protein hydrolysis and the release of bioactive peptides. Digested Ras cheese at 90 and 180 days of ripening displayed the highest bioactive peptides intensity. The variations in bioactive peptides amount during ripening and in vitro digestion were correlated with the changes in ACE-inhibitory, DPP-IV-inhibitory and antioxidant activities. The highest amounts of VPP and IPP were detected in digested Ras cheese at 90 days of ripening (17.44 and 36.50 mg/kg of cheese, respectively), whereas the highest concentrations of APFPE were found in undigested and digested 180-day ripened Ras cheese (82.09 and 52.01 mg/kg of cheese, respectively). The present investigation underlined potential differences in the biological effect after the ingestion of Ras cheese at different ripening times.

2023 - Impact of cooking methods of red-skinned onion on metabolic transformation of phenolic compounds and gut microbiota changes [Articolo su rivista]
Cattivelli, A.; Nissen, L.; Casciano, F.; Tagliazucchi, D.; Gianotti, A.
abstract

Herein, we investigated the stability and bioaccessibility of phenolics in differently cooked red-skinned onion (RSO) and consequently their impact on the gut microbiota and metabolism of phenolics. In fact, the different processes used to cook vegetables can modify and re-arrange the molecular profiles of bioactive compounds, such as phenolics in phenolic-rich vegetables, such as RSO. Fried and grilled RSO were compared to raw RSO and a blank control and subjected to oro-gastro-intestinal digestion and subsequent colonic fermentation. For upper gut digestion, the INFOGEST protocol was used, and for lower gut fermentation, a short-term batch model, namely, MICODE (multi-unit in vitro colon gut model), was employed. During the process, phenolic compound profile (through high-resolution mass spectrometry) and colon microbiomics (qPCR of 14 core taxa) analyses were performed. According to the results, the degradation driven by the colon microbiota of RSO flavonols resulted in the accumulation of three main metabolites, i.e., 3-(3 '-hydroxyphenyl)propanoic acid, 3-(3 '-hydroxyphenyl)acetic acid and 3-(3 ',4 '-dihydroxyphenyl)acetic acid. Also, colonic fermentation of raw onions resulted in a substantial increase in beneficial taxa, which was larger compared to the heat-treated onions, particularly Lactobacillales and beneficial clostridia. Also, a higher level of inhibition of opportunistic bacteria was seen for the raw onion samples, namely, Clostridium perfringens group and Escherichia coli. Thus, our results showed that RSO, and especially the raw one, is an excellent dietary source of flavonols that are strongly metabolized by gut bacteria and can positively modulate the gut microbiota. Although additional in vivo studies are necessary, this work is one of the first to explore how RSO processed with different cooking methods can differently impact the phenolic metabolism and microbiota composition in the large intestine of humans, fine-tuning the antioxidant nature of foods.

2023 - OeBAS and CYP716C67 catalyze the biosynthesis of health-beneficial triterpenoids in olive (Olea europaea) fruits [Articolo su rivista]
Alagna, F.; Reed, J.; Calderini, O.; Thimmappa, R.; Cultrera, N. G. M.; Cattivelli, A.; Tagliazucchi, D.; Mousavi, S.; Mariotti, R.; Osbourn, A.; Baldoni, L.
abstract

center dot The bioactive properties of olive (Olea europaea) fruits and olive oil are largely attributed to terpenoid compounds, including diverse triterpenoids such as oleanolic, maslinic and ursolic acids, erythrodiol, and uvaol. They have applications in the agri-food, cosmetics, and pharmaceutical industries. Some key steps involved in the biosynthesis of these compounds are still unknown.center dot Genome mining, biochemical analysis, and trait association studies have been used to identify major gene candidates controlling triterpenoid content of olive fruits.center dot Here, we identify and functionally characterize an oxidosqualene cyclase (OeBAS) required for the production of the major triterpene scaffold beta-amyrin, the precursor of erythrodiol, oleanolic and maslinic acids, and a cytochrome P450 (CYP716C67) that mediates 2 alpha oxidation of the oleanane- and ursane-type triterpene scaffolds to produce maslinic and corosolic acids, respectively. To confirm the enzymatic functions of the entire pathway, we have reconstituted the olive biosynthetic pathway for oleanane- and ursane-type triterpenoids in the heterologous host, Nicotiana benthamiana. Finally, we have identified genetic markers associated with oleanolic and maslinic acid fruit content on the chromosomes carrying the OeBAS and CYP716C67 genes.center dot Our results shed light on the biosynthesis of olive triterpenoids and provide new gene targets for germplasm screening and breeding for high triterpenoid content.

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.

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 - Fermentation of whey protein concentrate by Streptococcus thermophilus strains releases peptides with biological activities [Articolo su rivista]
Solieri, L.; Valentini, M.; Cattivelli, A.; Sola, L.; Helal, A.; Martini, S.; Tagliazucchi, D.
abstract

Cheese whey is the principal by-product of cheese making and is perceived as an environmental pollutant. Different technological procedures have been applied to convert whey into value-added products including whey protein concentrate (WPC). In the present study, 2 Lactobacillus helveticus strains and 5 Streptococcus thermophilus strains, isolated from natural whey starter used in Parmigiano Reggiano production, have been evaluated for their capacity to ferment WPC and to release bioactive peptides. WPC fermented with S. thermophilus RBC06 showed the highest antioxidant activity as well as angiotensin-converting enzyme- and dipeptidyl-peptidase-IV-inhibitory activities followed by WPC fermented by S. thermophilus RBC20 and RBN16. An untargeted peptidomics approach was applied to profile the peptides released after WPC fermentation. Several bioactive peptides, mainly angiotensin-converting enzyme inhibitors, were identified and the anti-hypertensive lactotripeptides valine-proline-proline (VPP) and isoleucine-proline-proline (IPP) were quantified. S. thermophilus RBC06 produced the highest amount of VPP (12.80 ± 0.45 mg/L) whereas the highest quantity of IPP (0.56 ± 0.02 mg/L) was detected in WPC fermented with S. thermophilus RBN16. The present study paved the way for the further exploitation of S. thermophilus RBC06 as new bioactive-peptide producer and for the application of WPC hydrolysates as additives for designing whey-based beverages with improved healthy properties.

2022 - In Vitro Bioaccessibility and Antioxidant Activity of Phenolic Compounds in Coffee-Fortified Yogurt [Articolo su rivista]
Helal, A.; Cattivelli, A.; Conte, A.; Tagliazucchi, D.
abstract

Yogurt is considered one of the most popular and healthy dairy products, and has been exploited as a delivery matrix for phenolic compounds. In this study, coffee powder was added to yogurt as a functional ingredient to produce coffee-fortified yogurt. Total phenolic compounds, antioxidant activity and individual hydroxycinnamic acids have been identified and quantified through mass spectrometry. The results from coffee-fortified yogurt were compared with fermented coffee and plain yogurt. Coffee-fortified yogurt had higher total phenolic content and antioxidant activity compared to plain yogurt. However, the total phenolic compounds found in coffee-fortified yogurt represented only 38.9% of the original content in coffee. Caffeoylquinic acids were the most abundant phenolic compounds in coffee. Fermented coffee and coffee-fortified yogurt displayed lower amounts of individual phenolic compounds with respect to coffee (69.8% and 52.4% of recovery, respectively). A protective effect of the yogurt matrix on total and individual coffee phenolic compounds has been observed after in vitro digestion, resulting in a higher bioaccessibility in comparison with digested fermented coffee. Moreover, coffee-fortified yogurt showed the highest antioxidant values after digestion. These findings clearly demonstrate that coffee-fortified yogurt can be considered a significant source of bioaccessible hydroxycinnamic acids, besides its health benefits as a fermented dairy product.

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

2022 - Milk-Derived Exosomes as Nanocarriers to Deliver Curcumin and Resveratrol in Breast Tissue and Enhance Their Anticancer Activity [Articolo su rivista]
González-Sarrías, Antonio; Iglesias-Aguirre, Carlos E; Cortés-Martín, Adrián; Vallejo, Fernando; Cattivelli, Alice; Del Pozo-Acebo, Lorena; Del Saz, Andrea; López de Las Hazas, María Carmen; Dávalos, Alberto; Espín, Juan Carlos
abstract

Dietary (poly)phenols are extensively metabolized, limiting their anticancer activity. Exosomes (EXOs) are extracellular vesicles that could protect polyphenols from metabolism. Our objective was to compare the delivery to breast tissue and anticancer activity in breast cancer cell lines of free curcumin (CUR) and resveratrol (RSV) vs. their encapsulation in milk-derived EXOs (EXO-CUR and EXO-RSV). A kinetic breast tissue disposition was performed in rats. CUR and RSV were analyzed using UPLC-QTOF-MS and GC-MS, respectively. Antiproliferative activity was tested in MCF-7 and MDA-MB-231 breast cancer and MCF-10A non-tumorigenic cells. Cell cycle distribution, apoptosis, caspases activation, and endocytosis pathways were determined. CUR and RSV peaked in the mammary tissue (41 +/- 15 and 300 +/- 80 nM, respectively) 6 min after intravenous administration of EXO-CUR and EXO-RSV, but not with equivalent free polyphenol concentrations. Nanomolar EXO-CUR or EXO-RSV concentrations, but not free CUR or RSV, exerted a potent antiproliferative effect on cancer cells with no effect on normal cells. Significant (p < 0.05) cell cycle alteration and pro-apoptotic activity (via the mitochondrial pathway) were observed. EXO-CUR and EXO-RSV entered the cells primarily via clathrin-mediated endocytosis, avoiding ATP-binding cassette transporters (ABC). Milk EXOs protected CUR and RSV from metabolism and delivered both polyphenols to the mammary tissue at concentrations compatible with the fast and potent anticancer effects exerted in model cells. Milk EXOs enhanced the bioavailability and anticancer activity of CUR and RSV by acting as Trojan horses that escape from cancer cells' ABC-mediated chemoresistance.

2022 - Roasting and frying modulate the phenolic profile of dark purple eggplant and differently change the colon microbiota and phenolic metabolites after in vitro digestion and fermentation in a gut model [Articolo su rivista]
Nissen, L.; Cattivelli, A.; Casciano, F.; Gianotti, A.; Tagliazucchi, D.
abstract

The way of cooking vegetables could differently affect the phenolic profiles of foods and their impact on human colon microbiota. In this work, we investigated the stability and bioaccessibility as well as the impact and fate of dark purple eggplant (DPE) phenolic compounds in the gut microbiota after grilling or frying in comparison to the raw one. After cooking, DPE underwent a gastro-intestinal digestion along with a proximal colon fermentation using the short-term batch model MICODE (multi-unit in vitro colon gut model). During the process, the phenolic compounds profiles (through high-resolution mass spectrometry) and microbiomics (qPCR of 14 core taxa) analyses were performed. Results showed that thermal treatments increased the amount of extractable phenolic compounds as well as their bioaccessibility. The highest gastro-intestinal release was observed in fried DPE (2468.46 ± 13.64 μmol/100 g), followed by grilled DPE (1007. 96 ± 12.84 μmol/100 g) and raw DPE (900.93 ± 10.56 μmol/100 g). Mass spectrometry analysis confirmed that colonic bacteria were able to metabolize DPE phenolic compounds mainly to 3-(3′-hydroxyphenyl)propanoic acid. Furthermore, results indicated that frying was better than grilling in terms of fostering more the growth of beneficial bacterial taxa and limiting that of opportunistic taxa. For example, fried DPE determined an increase in abundance of Bifidobacteriaceae Lactobacillales of 2.66 and 3.80 times. This work is one of the first exploring how cooking methods can affect the phenolic composition of DPE and differently impact on the colon microbiota tuning and modifying the food functionalities.

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 - 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 - 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.