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.

Sprouts of Moringa oleifera Lam.: Germination, Polyphenol Content and Antioxidant Activity.

(1) Background: In recent years, the consumption of sprouts, thanks to their high nutritional value, and the presence of bioactive compounds with antioxidant, antiviral and antibacterial properties, is becoming an increasingly widespread habit. Moringa oleifera Lam. (Moringa) seems to be an inexhaustible resource considering that many parts may be used as food or in traditional medicine; on the other hand, Moringa sprouts still lack a proper characterization needing further insights to envisage novel uses and applications. (2) Methods: In this study, a rapid and easy protocol to induce the in vivo and in vitro germination of Moringa seeds has been set up to obtain sprouts and cotyledons to be evaluated for their chemical composition. Moreover, the effects of sprouts developmental stage, type of sowing substrate, and gibberellic acid use on the chemical characteristics of extracts have been evaluated. (3) Results: Moringa seeds have a high germinability, both in in vivo and in vitro conditions. In addition, the extracts obtained have different total phenolic content and antioxidant activity. (4) Conclusions: This research provides a first-line evidence to evaluate Moringa sprouts as future novel functional food or as a valuable source of bioactive compounds.

Changes in chemical profile of Cascade hop cones according to the growing area.

BACKGROUND: The growing area has a substantial effect on plants, affecting secondary metabolism. For hops, different authors have studied the effect of growing area on the chemical composition of cones with the aim of verifying and understanding the changes in hop characters. Despite the scant literature the subject receives increasing attention by brewers and hop growers. The present study aimed to characterize, using gas chromatography-mass spectrometry (GC-MS), and high-performance liquid chromatography with ultraviolet detection (HPLC-UV), cones of hop (Humulus lupulus L.) cultivar Cascade. Plant material was obtained from nine different areas of Italy and compared with Cascade samples grown in the United States, Germany and Slovenia. RESULTS: Differences in bitter acids and xanthohumol content were observed. Nevertheless, no correlation between bitter acids and xanthohumol production, on the one hand, and rainfall, temperatures and latitude, on the other hand, were observed in our samples. The Slovenia samples were richer in molecules that confer hoppy, woody and flower notes; USA2 samples were more characterized by woody, earthy, grassy and floral aroma, quite different characters if compared to USA1, which had the lowest presence of grassy aromatic compounds. In the Italian samples, TRENTINO was the genotype most characterized by limonene presence. CONCLUSION: The results of this study are indicative of the importance for hop users to know and characterize hops coming from different growing regions. The study pays special attention to the characterization of the differences in chemical characters of Cascade hop in Italy, where hop cultivation has developed only recently, but is in continuous expansion. © 2019 Society of Chemical Industry.

Gliadin-mediated production of polyamines by RAW264.7 macrophages modulates intestinal epithelial permeability in vitro.

Celiac disease (CD) is an immune-mediated enteropathy sustained by dietary gluten in susceptible individuals, and characterized by a complex interplay between adaptive and innate responses against gluten peptides (PTG). In a recent contribution we have demonstrated that the treatment with PTG induces the expression and activity of arginase in both murine macrophages and human monocytes from healthy subjects, thus suggesting a role for arginine and its metabolites in gluten-triggered response of these cells. Here we further explore this field, by addressing the effects of PTG on polyamine synthesis and release in murine RAW264.7 macrophages, and how they affect epithelial permeability of Caco-2 monolayers. Results obtained show a massive production and release of putrescine by macrophages upon incubation with gluten peptides; this, in turn, causes a decrease in TEER in epithelial cells, indicating that PTG-driven secretion of polyamines by macrophages has a role in the modulation of intestinal permeability in vitro. At a molecular level, putrescine production appears referable to the activation of C/EBPß transcription factor, which is known to be responsible for arginase induction in activated macrophages and is a crucial mediator of inflammation. Whether these pathways are stimulated also in vivo deserves to be further investigated, as well as their role in gluten-driven cellular and intestinal defects typical of CD patients.

Phenolic and Volatile Composition of a Dry Spearmint (Mentha spicata L.) Extract.

The present paper reports a complete mass spectrometric characterization of both the phenolic and volatile fractions of a dried spearmint extract. Phenolic compounds were analysed by ultra-high performance liquid chromatography-electrospray ionization-mass spectrometry (UHPLC-ESI-MS(n)) and a total of 66 compounds were tentatively identified, being the widest phenolic characterisation of spearmint to date. The analysis suggests that the extract is composed of rosmarinic acid and its derivatives (230.5 ± 13.5 mg/g) with smaller amounts of salvianolic acids, caffeoylquinic acids, hydroxybenzoic acids, hydroxycinnamic acids, flavones, and flavanones. Head space solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) technique, that was applied to characterize the volatile fraction of spearmint, identified molecules belonging to different chemical classes, such as p-cymene, isopiperitone, and piperitone, dihydroedulan II, menthone, p-cymen-8-ol, and ß-linalool. This comprehensive phytochemical analysis can be useful to test the authenticity of this product rich in rosmarinic acid and other phenolics, and when assessing its biological properties. It may also be applied to other plant-derived food extracts and beverages containing a broad range of phytochemical compounds.

Phytochemical Profiling of Flavonoids, Phenolic Acids, Terpenoids, and Volatile Fraction of a Rosemary (Rosmarinus officinalis L.) Extract.

This paper presents a comprehensive analysis of the phytochemical profile of a proprietary rosemary (Rosmarinus officinalis L.) extract rich in carnosic acid. A characterization of the (poly)phenolic and volatile fractions of the extract was carried out using mass spectrometric techniques. The (poly)phenolic composition was assessed by ultra-high performance liquid chromatography-electrospray ionization-mass spectrometry (UHPLC-ESI-MSn) and a total of 57 compounds were tentatively identified and quantified, 14 of these being detected in rosemary extract for the first time. The rosemary extract contained 24 flavonoids (mainly flavones, although flavonols and flavanones were also detected), 5 phenolic acids, 24 diterpenoids (carnosic acid, carnosol, and rosmanol derivatives), 1 triterpenoid (betulinic acid), and 3 lignans (medioresinol derivatives). Carnosic acid was the predominant phenolic compound. The volatile profile of the rosemary extract was evaluated by head space solid-phase microextraction (HS-SPME) linked to gas chromatography-mass spectrometry (GC-MS). Sixty-three volatile molecules (mainly terpenes, alcohols, esters, aldehydes, and ketones) were identified. This characterization extends the current knowledge on the phytochemistry of Rosmarinus officinalis and is, to our knowledge, the broadest profiling of its secondary metabolites to date. It can assist in the authentication of rosemary extracts or rosemary-containing products or in testing its bioactivity. Moreover, this methodological approach could be applied to the study of other plant-based food ingredients.

Quantification of 3-MCPD and its mercapturic metabolite in human urine: validation of an LC-MS-MS method and its application in the general population.

A new method for the simultaneous quantitative determination in human urine of 3-monochloropropane-1,2-diol (3-MCPD), a toxic food contaminant, and its metabolite, 2,3-dihydroxypropyl mercapturic acid (DHPMA), was developed and validated. After urine dilution, the analytes were separated on an Atlantis®dC18 column and quantified by liquid chromatography-tandem mass spectrometry using isotopically labelled internal standards. The limits of quantification (S/N = 10) were 1.90 and 2.21 µg/L for 3-MCPD and DHPMA, respectively. Intra- and inter-day precision were lower than 6 % for each compound. Matrix effects were evaluated. Due to the high sensitivity and good accuracy of the method, 3-MCPD and DHPMA were found in 67 and 100 % of urine samples of healthy subjects, respectively.

Hydrolysed fumonisin B1 and N-(deoxy-D-fructos-1-yl)-fumonisin B1: stability and catabolic fate under simulated human gastrointestinal conditions.

Food processing may induce thermal degradation of fumonisins in corn via Maillard-type reactions, or alkaline hydrolysis via loss of the two tricarballylic acid moieties. In the former case, N-(1-deoxy-D-fructos-1-yl)-fumonisin B(1) (NDF) can be formed, while the latter derivative is called hydrolysed fumonisin B(1) (HFB(1)). The aim of this study was to deepen the knowledge about the gastrointestinal stability of HFB(1) and NDF in humans. Due to the lack of standard, NDF was chemically synthesised and cleaned up in high purity to be used for further experiments. While NDF is already partially cleaved (about 41%) during simulated digestion, it remained rather stable towards human colon microflora. In contrast to this, HFB(1) is partially metabolised by the colon microflora to unknown compounds after 24 h of fermentation, as seen by a loss of about 22%. Concluding, the cleavage of NDF during digestion as well as the likely metabolisation of HFB(1) emphasise the need for animal trials to ascertain their toxicity in vivo.

Short-term storage evaluation of quality and antioxidant capacity in chestnut-wheat bread.

BACKGROUND: Bread traditionally made from wheat is now often supplemented with alternative functional ingredients as chestnut flours; no data have been previously published about the staling of chestnut-containing bread. Thus short-term storage (3 days) for chestnut flour-supplemented soft wheat bread is evaluated by means of selected physicochemical properties (i.e. water dynamics, texture, colour, crumb grain characteristic, total antioxidant capacity). RESULTS: Bread prepared with a 20:80 ratio of chestnut:soft wheat flours maintained its moisture content in both crust and crumb. Crumb hardness, after baking, was found to be significantly higher than that of the soft wheat bread; it did not change during storage, whereas it significantly increased in the control bread until the end of the shelf life. The supplemented bread presented a heterogeneous crumb structure, with a significant decrease in the largest pores during shelf life, relative to the shrinkage of crumb grain. The control exhibited a significant redistribution of crumb holes, with a decrease in the smallest grain classes and an increase in the intermediate ones, most likely caused by cell wall thickening. The colour of the crumb remained unaltered in both breads. The crust of the control presented a significant decrease of a* (redness) and that of the supplemented bread exhibited a decrease of b* (yellowness). The antioxidant capacity was detected after day 1 of storage in the chestnut flour bread only. CONCLUSION: Chestnut flour supplementation could represent a feasible way of producing bread with improved characteristics, not only just after baking but also during shelf life.

Durum and soft wheat flours in sourdough and straight-dough bread-making.

In the present work, the bread-making performance of durum wheat flour under straight-dough and sourdough procedures were compared to those offered by soft wheat flour by means of selected physical properties (colour, texture, water dynamics, crumb grain characteristic, bulk volume) immediately after baking and during a 5-day shelf-life. The use of sourdough process better preserved both crumb grain characteristic and moisture content of the breads during shelf-life, independently of the wheat flour used. The flour seemed to significantly affect the water dynamics in sourdough breads, being the dehydration process of crust and under-crust faster in durum wheat breads. On the other hand, increasing trend of crumb firmness during the shelf-life was slower in durum wheat breads than in those obtained with soft wheat flour. Initial colour parameters of crust and crumb appeared to less change during shelf-life if durum wheat flour was used. Thus, the final quality of breads after baking and along the shelf-life was significantly affected by both the type of flours and the bread-making process. The results reported herein showed that technological performances of durum wheat flour, especially when combined with sourdough processes, could be successfully exploited for the production of innovative products in the bread-making industry.

Exploitation of Black Olive (Olea europaea L. cv. Piantone di Mogliano) Pomace for the Production of High-Value Bread.

In this study, the morpho-textural features, total phenolic content (TPC), and antioxidant capacity (AOC) of bread fortified with olive (Olea europaea L.) pomace were evaluated. Fresh olive pomace was subjected to microbiological and chemical (TPC, AOC, and fiber) analyses; then, the same olive pomace was analyzed during 1 to 6 months of storage at 4 °C or -20 °C. All olive pomace samples were used in 10%, 15%, or 20% amounts to produce type 0 soft wheat (Triticum aestivum) and whole wheat bread samples. The volatile organic compounds (VOCs) in the bread samples were also analyzed to assess the effect of the addition of the olive pomace on the flavor profile of the baked products. The TPC and AOC evaluation of olive pomace showed no differences among the analyzed samples (fresh, refrigerated, or frozen). Regarding the bread containing olive pomace, the specific volume was not affected by the amount or the storage methods of the added pomace. Bread samples produced with soft wheat flour showed the lowest hardness values relative to those produced with whole wheat flour, irrespective of the amount or storage method of the olive pomace. Regarding color, the crust and crumb of the bread samples containing 20% olive pomace were significantly darker. The bread samples containing 20% olive pomace had the highest TPC. The bread samples with fresh olive pomace were characterized by terpenoids, ketones, and aldehydes, whereas the bread samples containing refrigerated olive pomace were characterized by alcohols (mainly ethanol), acids, esters, and acetate. Finally, the bread samples with frozen olive pomace showed a volatile profile similar to that of bread produced with fresh olive pomace. Olive pomace was shown to be a suitable ingredient for producing bread with high nutritional value.

Masked mycotoxins are efficiently hydrolyzed by human colonic microbiota releasing their aglycones.

Fusarium mycotoxins are secondary metabolites produced by Fusarium spp. in cereals. Among them, deoxynivalenol (DON) and zearalenone (ZEN) are widespread worldwide contaminants of cereal commodities and are ranked as the most important chronic dietary risk factors. Their conjugates, known as masked mycotoxins, have been described but are still not accounted for in risk assessment studies. This study demonstrates for the first time that DON and ZEN are effectively deconjugated by the human colonic microbiota, releasing their toxic aglycones and generating yet unidentified catabolites. For this reason, masked mycotoxins should be considered when evaluating population exposure.

Towards Sustainable and Nutritionally Enhanced Flatbreads from Sprouted Sorghum, Tapioca, and Cowpea Climate-Resilient Crops.

This study aimed to develop high-quality flatbreads for low-income countries by using composite flours from climate-resilient crops, i.e., sprouted sorghum, tapioca, and cowpea, as partial alternatives to imported wheat. Through the experimental design, several flatbread prototypes were developed that maximized the content of sprouted sorghum and cowpea flours and minimized the content of wholewheat flour. Three of them were chosen based on the best textural, nutritional (highest intake of energy, proteins, and micronutrients-iron, zinc and vitamin A), and economic (cheapest in Sierra Leone, Tanzania, Burundi, and Togo) features. The physicochemical properties, in vitro starch digestibility, total phenolic content, antioxidant capacity, and sensory acceptability were also measured for the samples. The experimental flatbreads showed lower rapidly digestible starch and higher resistant starch contents than the control (100% wholewheat based), and were also richer in phenolic content and higher in antioxidant activity. Moreover, one of the prototypes was perceived to be as acceptable as the control for texture and flavour properties. The ranking test, performed after explaining the nature of the samples, revealed that the flatbread meeting the nutritional criteria was the preferred one. Overall, the use of composite flour from climate-resilient crops was proven to be an efficient strategy to obtain high-quality flatbread.

Lacticaseibacillus Strains Isolated from Raw Milk: Screening Strategy for Their Qualification as Adjunct Culture in Cheesemaking.

The microbial ecology fundamentals of raw milk and long-ripened cheeses consist of a complex interaction between starter lactic acid bacteria (SLAB) and non-starter LAB (NSLAB). Although NSLAB aromatic properties are paramount, other phenotypic traits need to be considered for their use as adjunct cultures, such as the capability to endure technological parameters encountered during cheesemaking. The present study focused on the isolation and characterization of NSLAB from spontaneously fermented raw cow's milk coming from 20 dairies that produce Grana Padano PDO cheese. From 122 isolates, the screening process selected the 10 most diverse strains belonging to Lacticaseibacillus spp. to be phenotypically characterized. The strains were tested for their growth performance in milk in combination with the application of technological stresses, for their ability to produce volatile compounds after their growth in milk, and for their ability to use different nutrient sources and resist chemicals. The complex characterization qualified the strains 5959_Lbparacasei and 5296_Lbparacasei as the best candidates to be used as adjunct strains in the production of raw milk and long-ripened cheeses, provided that antibiotic resistance is measured before their employment. Other strains with interesting aromatic capabilities but lower heat resistance were 5293_Lbparacasei, 5649_Lbparacasei and 5780_Lbparacasei, which could be candidates as adjunct strains for uncooked cheese production.

Perenniality, more than genotypes, shapes biological and chemical rhizosphere composition of perennial wheat lines.

Perennial grains provide various ecosystem services compared to the annual counterparts thanks to their extensive root system and permanent soil cover. However, little is known about the evolution and diversification of perennial grains rhizosphere and its ecological functions over time. In this study, a suite of -OMICSs - metagenomics, enzymomics, metabolomics and lipidomics - was used to compare the rhizosphere environment of four perennial wheat lines at the first and fourth year of growth in comparison with an annual durum wheat cultivar and the parental species Thinopyrum intermedium. We hypothesized that wheat perenniality has a greater role in shaping the rhizobiome composition, biomass, diversity, and activity than plant genotypes because perenniality affects the quality and quantity of C input - mainly root exudates - hence modulating the plant-microbes crosstalk. In support of this hypothesis, the continuous supply of sugars in the rhizosphere along the years created a favorable environment for microbial growth which is reflected in a higher microbial biomass and enzymatic activity. Moreover, modification in the rhizosphere metabolome and lipidome over the years led to changes in the microbial community composition favoring the coexistence of more diverse microbial taxa, increasing plant tolerance to biotic and abiotic stresses. Despite the dominance of the perenniality effect, our data underlined that the OK72 line rhizobiome distinguished from the others by the increase in abundance of Pseudomonas spp., most of which are known as potential beneficial microorganisms, identifying this line as a suitable candidate for the study and selection of new perennial wheat lines.

A Computational Understanding of Inter-Individual Variability in CYP2D6 Activity to Investigate the Impact of Missense Mutations on Ochratoxin A Metabolism.

Cytochrome P-450 (CYP) enzymes have a key role in the metabolism of xenobiotics of food origin, and their highly polymorphic nature concurs with the diverse inter-individual variability in the toxicokinetics (TK) and toxicodynamics (TD) of food chemicals. Ochratoxin A is a well-known mycotoxin which contaminates a large variety of food and is associated with food safety concerns. It is a minor substrate of CYP2D6, although the effects of CYP2D6 polymorphisms on its metabolism may be overlooked. Insights on this aspect would provide a useful mechanistic basis for a more science-based hazard assessment, particularly to integrate inter-individual differences in CYP2D6 metabolism. This work presents a molecular modelling approach for the analysis of mechanistic features with regard to the metabolic capacity of CYP2D6 variants to oxidise a number of substrates. The outcomes highlighted that a low-frequency CYP2D6 variant (CYP2D6*110) is likely to enhance ochratoxin A oxidation with possible consequences on TK and TD. It is therefore recommended to further analyse such TK and TD consequences. Generally speaking, we propose the identification of mechanistic features and parameters that could provide a semi-quantitative means to discriminate ligands based on the likelihood to undergo transformation by CYP2D6 variants. This would support the development of a fit-for-purpose pipeline which can be extended to a tool allowing for the bulk analysis of a large number of compounds. Such a tool would ultimately include inter-phenotypic differences of polymorphic xenobiotic-metabolising enzymes in the hazard assessment and risk characterisation of food chemicals.

Assessing food authenticity through protein and metabolic markers.

This chapter aims to address an issue of ancient origins, but more and more topical in a globalized world in which consumers and stakeholders are increasingly aware: the authenticity of food. Foods are systems that can also be very complex, and verifying the correspondence between what is declared and the actual characteristics of the product is often a challenging issue. The complexity of the question we want to answer (is the food authentic?) means that the answer is equally articulated and makes use of many different analytical techniques. This chapter will consider the chemical analyses of foods aimed at guaranteeing their authenticity and will focus on frontier methods that have been developed in recent years to address the need to respond to ever-increasing guarantees of authenticity. Targeted and non-targeted approaches will be considered for verifying the authenticity of foods, through the study of different classes of constituents (proteins, metabolites, lipids, flavors). The numerous approaches available (proteomics, metabolomics, lipidomics) and the related analytical techniques (LC-MS, GC-MS, NMR) are first described from a more general point of view, after which their specific application for the purposes of authentication of food is addressed.

Effects of Thermal and High-Pressure Processing on Quality Features and the Volatile Profiles of Cloudy Juices Obtained from Golden Delicious, Pinova, and Red Delicious Apple Cultivars.

In this study, juices extracted from three apple cultivars (Golden Delicious, Pinova, and Red Delicious) were stabilized by means of thermal treatment (TT) and high-pressure processing (HPP, 600 MPa 3 min); pH, total titratable acidity, total soluble solids content, color, and viscosity, as well as volatile profile, were investigated. Qualitative characteristics (pH, titratable acidity, colorimetric parameters, viscosity, and volatile profile) results were significantly influenced by both cultivars and treatments; for example, juice viscosity greatly increased after HPP treatment for Golden Delicious, and after both TT and HPP for Pinova, while no influence of stabilization treatment was registered for Red Delicious juices. Regarding the volatile profile, for Golden Delicious cultivar, HPP treatment determined an increase in volatile compounds for most of the classes considered, leading to a supposed quality implementation. For the other two cultivars, the stabilization treatment that better preserved the volatile profile was the HPP one, even if the results were quite similar to the thermal treatment. Further studies are needed to evaluate different time/pressure combinations that could give better results, depending on the specific apple cultivar.

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.

Characterization of a potential nutraceutical ingredient: pomegranate (Punica granatum L.) seed oil unsaponifiable fraction.

The chemical fingerprinting of the unsaponifiable fraction of different Punica granatum seed oils was performed in order to evaluate their potential as a functional food ingredient. Qualitative and quantitative determinations of tocopherol, aliphatic alcohol (including policosanol), squalene, phytosterols and triterpene contents were performed by GC-MS. A high yield (3.1-4.2%) of unsaponifiable matter was obtained and consistent levels of squalene (up to 800 mg/kg) and policosanol (118-185 mg/kg) were noticed. ß-sitosterol (up to 8069 mg/kg) and cycloartenol (5916-7766 mg/kg) were predominant in phytosterol and triterpene fractions, while ß- and δ-tocopherol were the most abundant vitamin E forms. Some minor variations were noticed between samples. From the results obtained, it can be suggested that the seed oil of P. granatum can be considered an interesting alimentary source of substances of nutraceutical value involved in the modulation of cholesterol metabolism.