Virtual display of targets: A new level to rise the current understanding of ochratoxin A toxicity from a molecular standpoint.

Ochratoxin A (OTA) is a mycotoxin spread worldwide contaminating several food and feed commodities and rising concerns for humans and animals. OTA toxicity has been thoroughly assessed over the last 60 years revealing a variety of adverse effects, including nephrotoxicity, hepatotoxicity and possible carcinogenicity. However, the underpinning mechanisms of action have yet to be completely displayed and understood. In this framework, we applied a virtual pipeline based on molecular docking, dynamics and umbrella simulations to display new OTA potential targets. The results collected consistently identified OGFOD1, a key player in protein translation, as possibly inhibited by OTA and its 2'R diastereomer. This is consistent with the current knowledge of OTA's molecular toxicology and may fill some gaps from a mechanistic standpoint. This could pave the way for further dedicated analysis focusing their attention on the OTA-OGFOD1 interaction, expanding the current understanding of OTA toxicity at a molecular level.

A mechanistic toxicology study to grasp the mechanics of zearalenone estrogenicity: Spotlighting aromatase and the effects of its genetic variability.

Zearalenone (ZEN) is a mycoestrogen produced by Fusarium fungi contaminating cereals and in grain-based products threatening human and animal health due to its endocrine disrupting effects. Germane to the mechanisms of action, ZEN may activate the estrogen receptors and inhibit the estrogens-producing enzyme aromatase (CYP19A1). Both show single nucleotide variants (SNVs) among humans associated with a diverse susceptibility of being activated or inhibited. These variations might modify the endocrine disrupting action of ZEN, requiring dedicated studies to improve its toxicological understanding. This work focused on human aromatase investigating via 3D molecular modelling whether some of the SNVs reported so far (n = 434) may affect the inhibitory potential of ZEN. It has been also calculated the inhibition capability of α-zearalenol, the most prominent and estrogenically potent phase I metabolite of ZEN, toward those aromatase variants with an expected diverse sensitivity of being inhibited by ZEN. The study: i) described SNVs likely associated with a different susceptibility to ZEN and α-zearalenol inhibition - like T310S that is likely more susceptible to inhibition, or D309G and S478F that are possibly inactive variants; ii) proofed the possible existence of inter-individual susceptibility to ZEN; iii) prioritized aromatase variants for future investigations toward a better comprehension of ZEN xenoestrogenicity at an individual level.

Untargeted Metabolomics of Meat Digests: Its Potential to Differentiate Pork Depending on the Feeding Regimen.

Meat quality seems to be influenced by the dietary regimes applied for animal feeding. Several research studies are aimed at improving meat quality, preserving it from oxidative processes, by the incorporation of antioxidant components in animal feeding. The main part of these studies evaluates meat quality, determining different parameters directly on meat, while few research studies take into account what may happen after meat ingestion. To address this topic, in this study, an in vitro gastrointestinal digestion protocol was applied to two different pork muscles, longissimus dorsi and rectus femoris, obtained from pigs fed with different diets. In detail, two groups of 12 animals each were subjected to either a conventional diet or a supplemented diet with extruded linseeds as a source of omega-3 fatty acids and plant extracts as a source of phenolics antioxidant compounds. The digested meat was subjected to an untargeted metabolomics approach. Several metabolites deriving from lipid and protein digestion were detected. Our untargeted approach allowed for discriminating the two different meat cuts, based on their metabolomic profiles. Nonetheless, multivariate statistics allowed clearly discriminating between samples obtained from different animal diets. In particular, the inclusion of linseeds and polyphenols in the animal diet led to a decrease in metabolites generated from oxidative degradation reactions, in comparison to the conventional diet group. In the latter, fatty acyls, fatty aldehydes and oxylipins, as well as cholesterol and vitamin D3 precursors and derivatives, could be highlighted.

An In Silico Framework to Mine Bioactive Peptides from Annotated Proteomes: A Case Study on Pancreatic Alpha Amylase Inhibitory Peptides from Algae and Cyanobacteria.

Bioactive peptides may exert beneficial activities in living organisms such as the regulation of glucose metabolism through the inhibition of alpha amylases. Algae and cyanobacteria are gaining a growing interest for their health-promoting properties, and possible effects on glucose metabolism have been described, although the underlying mechanisms need clarification. This study proposes a computer-driven workflow for a proteome-wide mining of alpha amylase inhibitory peptides from the proteome of Chlorella vulgaris, Auxenochlorella protothecoides and Aphanizomenon flos-aquae. Overall, this work presents an innovative and versatile approach to support the identification of bioactive peptides in annotated proteomes. The study: (i) highlighted the presence of alpha amylase inhibitory peptides within the proteomes under investigation (including ELS, which is among the most potent inhibitory tripeptides identified so far); (ii) mechanistically investigated the possible mechanisms of action; and (iii) prioritized further dedicated investigation on the proteome of C. vulgaris and A. flos-aquae, and on CSSL and PGG sequences.

Cleaning the Label of Cured Meat; Effect of the Replacement of Nitrates/Nitrites on Nutrients Bioaccessibility, Peptides Formation, and Cellular Toxicity of In Vitro Digested Salami.

Curing salts composed of mixtures of nitrates and nitrites are preservatives widely used in processed meats. Despite many desirable technological effects, their use in meat products has been linked to methemoglobinemia and the formation of nitrosamines. Therefore, an increasing "anti-nitrite feeling" has grown among meat consumers, who search for clean label products. In this view, the use of natural compounds as alternatives represents a challenge for the meat industry. Processing (including formulation and fermentation) induces chemical or physical changes of food matrix that can modify the bioaccessibility of nutrients and the formation of peptides, impacting on the real nutritional value of food. In this study we investigated the effect of nitrate/nitrite replacement with a combination of polyphenols, ascorbate, and nitrate-reducing microbial starter cultures on the bioaccessibility of fatty acids, the hydrolysis of proteins and the release of bioactive peptides after in vitro digestion. Moreover, digested salami formulations were investigated for their impacts on cell proliferation and genotoxicity in the human intestinal cellular model (HT-29 cell line). The results indicated that a replacement of synthetic nitrates/nitrites with natural additives can represent a promising strategy to develop innovative "clean label" salamis without negatively affecting their nutritional value.

Mechanistic Insights into Angiotensin I-Converting Enzyme Inhibitory Tripeptides to Decipher the Chemical Basis of Their Activity.

Food proteins are an important source of bioactive peptides, and the angiotensin I-converting enzyme (ACE) inhibitors are worthy of attention for their possible beneficial effects in subjects with mild hypertension. However, the chemical basis underpinning their activity is not well-understood, hampering the discovery of novel inhibitory sequences from the plethora of peptides encrypted in food proteins. This work combined computational and in vitro investigations to describe precisely the chemical basis of potent inhibitory tripeptides. A substantial set of previously uncharacterized tripeptides have been investigated in silico and in vitro, and LCP was described for the first time as a potent ACE inhibitory peptide with IC50 values of 8.25 and 6.95 µM in cell-free and cell-based assays, respectively. The outcomes presented could serve to better understand the chemical basis of already characterized potent inhibitory tripeptides or as a blueprint to design novel and potent inhibitory peptides and peptide-like molecules.

A molecular insight into the lipid changes of pig Longissimus thoracis muscle following dietary supplementation with functional ingredients.

In this work, the Longissimus thoracis pig skeletal muscle was used as a model to investigate the impact of two different diets, supplemented with n-3 polyunsaturated fatty acids from extruded linseed (L) and polyphenols from grape skin and oregano extracts (L+P), on the lipidomic profile of meat. A standard diet for growing-finishing pigs (CTRL) was used as a control. Changes in lipids profile were investigated through an untargeted lipidomics and transcriptomics combined investigation. The lipidomics identified 1507 compounds, with 195 compounds fitting with the MS/MS spectra of LipidBlast database. When compared with the CTRL group, the L+P diet significantly increased 15 glycerophospholipids and 8 sphingolipids, while the L diet determined a marked up-accumulation of glycerolipids. According to the correlations outlined between discriminant lipids and genes, the L diet may act preventing adipogenesis and the related inflammation processes, while the L+P diet promoted the expression of genes involved in lipids' biosynthesis and adipogenic extracellular matrix formation and functioning.

A heuristic, computer-driven and top-down approach to identify novel bioactive peptides: A proof-of-principle on angiotensin I converting enzyme inhibitory peptides.

Bioactive peptides are short peptides (3-20 amino acid residues in length) endowed of specific biological activities. The identification and characterization of bioactive peptides of food origin are crucial to better understand the physiological consequences of food, as well as to design novel foods, ingredients, supplements, and diets to counteract mild metabolic disorders. For this reason, the identification of bioactive peptides is also relevant from a pharmaceutical standpoint. Nevertheless, the systematic identification of bioactive sequences of food origin is still challenging and relies mainly on the so defined "bottom-up" approaches, which rarely results in the total identification of most active sequences. Conversely, "top-down" approaches aim at identifying bioactive sequences with certain features and may be more suitable for the precise identification of very potent bioactive peptides. In this context, this work presents a top-down, computer-assisted and hypothesis-driven identification of potent angiotensin I converting enzyme inhibitory tripeptides, as a proof of principle. A virtual library of 6840 tripeptides was screened in silico to identify potential highly potent inhibitory peptides. Then, computational results were confirmed experimentally and a very potent novel sequence, LMP was identified. LMP showed an IC50 of 15.8 and 6.8 µM in cell-free and cell-based assays, respectively. In addition, a bioinformatics approach was used to search potential food sources of LMP. Yolk proteins were identified as a possible relevant source to analyze in further experiments. Overall, the method presented may represent a powerful and versatile framework for a systematic, high-throughput and top-down identification of bioactive peptides.

Effect of fermentation with single and co-culture of lactic acid bacteria on okara: evaluation of bioactive compounds and volatile profiles.

Okara is the main soybean by-product resulting from the processing of soy milk and tofu. Despite being a product with a lot of potential and rich in many bioactive compounds such as polyphenols, it presents an unpleasant, rancid aroma. For this reason its use in the food industry is limited. In this study, we have reported the integral use of okara in a solid state fermentation process, conducted with wild strains of lactic acid bacteria, to evaluate the effect of bacterial metabolism on the volatile and polyphenolic profiles. Strains belonging to Lactobacillus acidophilus, Lacticaseibacillus rhamnosus and Pediococcus acidilactici species were used in monoculture and, for the first time, in co-culture. The results showed an improvement in the aromatic fraction showing a decrease in hexanal, responsible for off-flavour, and an increase in ketones with fruity and buttery notes in fermented okara. Polyphenols were also affected, and, in particular, a bioconversion of glucoside isoflavones to the aglycone forms was highlighted in all fermented substrates. In addition, the appearance of both phenyllactic and p-hydroxyphenyllactic acids as well as the increase in indole-3-lactic acid was observed for the first time upon okara fermentation. Overall, the co-culture appears to be the most promising for biovalorization of okara, thereby opening the possibility of its use in the development of functional ingredients.

A Hybrid In Silico/In Vitro Target Fishing Study to Mine Novel Targets of Urolithin A and B: A Step Towards a Better Comprehension of Their Estrogenicity.

SCOPE: Urolithin A and B are gut metabolites of ellagic acid and ellagitannins associated with many beneficial effects. Evidence in vitro pointed to their potential as estrogenic modulators. However, both molecular mechanisms and biological targets involved in such activity are still poorly characterized, preventing a comprehensive understanding of their bioactivity in living organisms. This study aimed at rationally identifying novel biological targets underlying the estrogenic-modulatory activity of urolithins. METHODS AND RESULTS: The work relies on an in silico/in vitro target fishing study coupling molecular modeling with biochemical and cell-based assays. Estrogen sulfotransferase and 17ß-hydroxysteroid dehydrogenase are identified as potentially subject to inhibition by the investigated urolithins. The inhibition of the latter undergoes experimental confirmation either in a cell-free or cell-based assay, validating computational outcomes. CONCLUSIONS: The work describes target fishing as an effective tool to identify unexpected targets of food bioactives detailing the interaction at a molecular level. Specifically, it described, for the first time, 17ß-hydroxysteroid dehydrogenase as a target of urolithins and highlighted the need of further investigations to widen the understanding of urolithins as estrogen modulators in living organisms.

Alternaria toxins as casein kinase 2 inhibitors and possible consequences for estrogenicity: a hybrid in silico/in vitro study.

Emerging mycotoxins produced by Alternaria spp. were previously reported to exert cytotoxic, genotoxic, but also estrogenic effects in human cells. The involved mechanisms are very complex and not fully elucidated yet. Thus, we followed an in silico target fishing approach to extend knowledge on the possible biological targets underlying the activity of alternariol, taken as the signature compound of Alternaria toxins. Combining ligand-based screening and structure-based modeling, the ubiquitous casein kinase 2 (CK2) was identified as a potential target for the compound. This result was validated in a cell-free in vitro CK2 activity assay, where alternariol inhibited CK2 with an IC50 of 707 nM. As CK2 was recently discussed to influence estrogen receptor (ER) transcription and DNA-binding affinity, we assessed a potential impact on the mRNA levels of ERα or ERß by qRT-PCR and on nuclear localization of the receptors by confocal microscopy, using estrogen-sensitive Ishikawa cells as a model. While AOH did not affect the transcription of ERα or ERß, an increase in nuclear localization of ERα after incubation with 10 µM AOH was observed. However, this effect might be due to ER binding affinity and therefore estrogenicity of AOH. Furthermore, in silico docking simulation revealed not only AOH, but also a number of other Alternaria toxins as potential inhibitors of CK2, including alternariol monomethyl ether and the perylene quinone derivative altertoxin II (ATX-II). These findings were representatively confirmed in vitro for the perylene quinone derivative altertoxin II, which was found to inhibit the kinase with an IC50 of 5.1 µM. Taken together, we propose CK2 inhibition as an additional mechanism to consider in future studies for alternariol and several other Alternaria toxins.

Assessment of the Multifunctional Behavior of Lupin Peptide P7 and Its Metabolite Using an Integrated Strategy.

LTFPGSAED (P7) is a multifunctional hypocholesterolemic and hypoglycemic lupin peptide. While assessing its angiotensin-converting enzyme (ACE) inhibitory activity, it was more effective in intestinal Caco-2 cells (IC50 of 13.7 µM) than in renal HK-2 cells (IC50 of 79.6 µM). This discrepancy was explained by the metabolic transformation mediated by intestinal peptidases, which produced two main detected peptides, TFPGSAED and LTFPG. Indeed LTFPG, dynamically generated by intestinal dipeptidyl peptidase IV as well as its parent peptide P7 were linearly absorbed by mature Caco-2 cells. An in silico study demonstrated that the metabolite was a better ligand of the ACE enzyme than P7. These results are in agreement with an in vivo study, previously performed by Aluko et al., which has shown that LTFPG is an effective hypotensive peptide. Our work highlights the dynamic nature of bioactive food peptides that may be modulated by the metabolic activity of intestinal cells.

"Bottom-Up" Strategy for the Identification of Novel Soybean Peptides with Angiotensin-Converting Enzyme Inhibitory Activity.

IAVPTGVA (Soy1) and LPYP are two soybean peptides, which display a multifunctional behavior, showing in vitro hypocholesterolemic and hypoglycemic activities. A preliminary screening of their structures using BIOPEP suggested that they might be potential angiotensin-converting enzyme (ACE) inhibitors. Therefore, a bottom-up-aided approach was developed in order to clarify the in vitro hypotensive activity. Soy1 and LPYP dropped the intestinal and renal ACE enzyme activity with IC50 values equal to 14.7 ± 0.28 and 5.0 ± 0.28 µM (Caco-2 cells), and 6.0 ± 0.35 and 6.8 ± 0.20 µM (HK-2 cells), respectively. In parallel, a molecular modeling study suggested their capability to act as competitive inhibitors of this enzyme. Finally, in order to increase both their stability and hypotensive properties, a suitable strategy for the harmless control of their release from a nanomaterial was developed through their encapsulation into the RADA16-assembling peptide.

An in silico structural approach to characterize human and rainbow trout estrogenicity of mycotoxins: Proof of concept study using zearalenone and alternariol.

The mycotoxins zearalenone and alternariol may contaminate food and feed raising toxicological concerns due to their estrogenicity. Inter-species differences in their toxicokinetics and toxicodynamics may occur depending on evolution of taxa-specific traits. As a proof of principle, this manuscript investigates the comparative toxicodynamics of zearalenone, its metabolites (alpha-zearalenol and beta-zearalenol), and alternariol with regards to estrogenicity in humans and rainbow trout. An in silico structural approach based on docking simulations, pharmacophore modeling and molecular dynamics was applied and computational results were analyzed in comparison with available experimental data. The differences of estrogenicity among species of zearalenone and its metabolites have been structurally explained. Also, the low estrogenicity of alternariol in trout has been characterized here for the first time. This approach can provide a powerful tool for the characterization of interspecies differences in mycotoxin toxicity for a range of protein targets and relevant compounds for the food- and feed-safety area.

The impact of processing on the phenolic acids, free betaine and choline in Triticum spp. L. whole grains and milling by-products.

Wheat (Triticum spp. L.) is an important source of nutrients and bioactive compounds with recognized beneficial effects. Wheat undergoes several processes with the final aim of separating the endosperm from the outer layers, usually discarded. In this study, free and bound phenolic acids (PAs) profile, betaine and choline contents were quantified in six different wheat species (durum and bread wheat, turanicum wheat, einkorn, emmer and spelt), the corresponding milling by-products (bran, middlings, aleurone and I, II and III steps of debranning) and flour/semolina, using UHPLC-MS/MS methods. The bound form of phenolics was the component present in higher concentration (80% of the total, in average) and ferulic acid was the most abundant compounds, representing between 67 and 73 % of total PAs. Among the species, bread wheat grain totalized the highest content of total PAs (1209.31 ± 7.3 µg g-1 d.w.). Betaine and choline are abundantly present in wheat species. In general, the highest content of bioactive compounds was found in bran (3 times higher than whole grains), emphasizing the good nutritional profile of these by-products. The milling process leads to a severe reduction of phenolic acids and methyl-donors in the end-products.

Evaluation of polyphenolic compounds in membrane concentrated pistachio hull extract.

Pistachio hull is a substantial source of natural polyphenols, but a substantial volume is being wasted annually. Aqueous pistachio green hull extract (PGHE) was subjected to two-stage membrane process in order to separate a polyphenol rich fraction. The membrane conditions of each stage were investigated. Total phenolic content and antioxidant activity were determined by Folin-Ciocalteu and DPPH· assay; also membrane fouling was monitored. The use of the 1 kDa cellulose membrane accompanied by 4 bar pressure and 250 rpm stirring speed was observed to be successful in the separation of a fraction with the highest amount of phenolic compound and antioxidant activity, in the retentate part. UHPLC/MSn characterization of PGHE polyphenols enabled us to identify 34 compounds, including the most abundant galloylshikimic acids, gallic acid, theogallin, galloyl-O-hexoside, quercetin-O-hexoside and pyrogallol.

In vitro metabolism of elderberry juice polyphenols by lactic acid bacteria.

In this study, ten strains of Lactobacillus were used to assess the in vitro metabolism of elderberry juice polyphenols. Total polyphenolic compounds increased after starter addition, especially with three L. rhamnosus and one L. plantarum strains, of dairy origin: quercetin-3-O-rutinoside was the most abundant compound (from 39.02 ±â€¯5.28 to 127.56 ±â€¯11.34 µg/mL) and hydroxycinnamic acids, flavonols and anthocyanins reached the highest amounts. When L. plantarum were used phenyllactic acids presented a value of 7.05 ±â€¯2.38 µg/mL, while in the other samples it was around 5.56 ±â€¯1.65 µg/mL. Hydroxycinnamic and hydroxybenzoic acids were subjected to lactic acid bacteria metabolism: caffeic and protocatechuic acids were consumed during fermentation while dihydrocaffeic acid and catechol were produced. Anthocyanins increased in a strain-specific way. So, by this study we highlighted that dairy strains can produced (phenyllactic acids), modified (hydroxycinnamic acids) or increased (flavonols glycosides and anthocyanins) phenolic compounds.

The Influence of Viable Cells and Cell-Free Extracts of Lactobacillus casei on Volatile Compounds and Polyphenolic Profile of Elderberry Juice.

In this study, four strains of Lactobacillus casei, as viable cells or cell-free extracts (CFE), were added to elderberry juice in order to evaluate their effect on phenolic and aromatic profile. Two of them were able to grow in juice while the others showed zero-growth. The same strains were lysed and added as extracts in elderberry juice. Multivariate statistical analysis show a separation among samples containing growing cells, non-growing cells, CFE, highlighting the particularities of specific strains. Juices added with CFE presented the highest amount of esters. The strains showing growth phenotype cause an increase of phenyllactic acids. The highest concentration of volatile compounds, particularly of alcohols, terpenes and norisoprenoids (responsible for typical elderberry notes) was observed in samples with strains showing zero-growth. Moreover, a significant increase in anthocyanin content was observed in these samples, suggesting the possible use of Lactobacillus for increasing specific molecules, even for non-multiplying bacterial cell. Considering that this is the first study concerning the use of non-growing cells in fruit juice, the potential of strains is still to be explored and it may have a significant technological application in the development of a microbial collection useful for fruit juice industry.

On the Mechanism of Action of Anti-Inflammatory Activity of Hypericin: An In Silico Study Pointing to the Relevance of Janus Kinases Inhibition.

St. John's Wort (Hypericum perforatum L.) flowers are commonly used in ethnomedical preparations with promising outcomes to treat inflammation both per os and by topical application. However, the underlying molecular mechanisms need to be described toward a rational, evidence-based, and reproducible use. For this purpose, the aptitude of the prominent Hypericum metabolite hypericin was assessed, along with that of its main congeners, to behave as an inhibitor of janus kinase 1, a relevant enzyme in inflammatory response. It was used a molecular modeling approach relying on docking simulations, pharmacophoric modeling, and molecular dynamics to estimate the capability of molecules to interact and persist within the enzyme pocket. Our results highlighted the capability of hypericin, and some of its analogues and metabolites, to behave as ATP-competitive inhibitor providing: (i) a likely mechanistic elucidation of anti-inflammatory activity of H. perforatum extracts containing hypericin and related compounds; and (ii) a rational-based prioritization of H. perforatum components to further characterize their actual effectiveness as anti-inflammatory agents.

Occurrence of non-proteolytic amino acyl derivatives in dry-cured ham.

Proteolysis is the most important event occurring during maturation of dry-cured hams: it strongly influences the flavour and the texture of the aged ham by the accumulation of peptides and free amino acids released by protein hydrolysis. Apart from compounds of proteolytic origin, it has been demonstrated that also non-proteolytic amino acyl derivatives (γ-glutamyl amino acids, pyroglutamyl-amino acids and lactoyl-amino acids) may accumulate during ripening of cheese, and they can be also found in fermented soy sauce, where they contribute to the umami taste of the products. Using a semi-quantitative analysis, in this paper we report the occurrence of significant amounts of γ-glutamyl amino acids and, for the first time, pyroglutamyl-amino acids and lactoyl-amino acids, in aged ham. The amino acid counterparts were mainly found to be hydrophobic amino acids. The amount of these compounds was found to increase with time, because they are not degraded by proteolytic activity. They were also found to be stable to simulated gastrointestinal digestion. Angiotensin Converting Enzyme inhibitory activity was also tested, but they were not found to be characterized by significant ACE-inhibitory activity.