Pomegranate supplementation alleviates dyslipidemia and the onset of non-alcoholic fatty liver disease in Wistar rats by shifting microbiota and producing urolithin-like microbial metabolites.

Non-alcoholic fatty liver disease (NAFLD), obesity and related chronic diseases are major non-communicable diseases with high mortality rates worldwide. While dietary sugars are known to be responsible for insulin resistance and metabolic syndrome (MetS), the underlying pathophysiological effects of sustained fructose consumption require further elucidation. We hypothesize that certain bioactive compounds (i.e. punicalagin and ellagic acid) from dietary pomegranate could counteract the harmful effects of sustained fructose consumption in terms of obesity and liver damage. The present study aimed to elucidate both the molecular mechanisms involved in the pathophysiology associated with fructose intake and the effect of a punicalagin-rich commercial pomegranate dietary supplement (P) used as a nutritional strategy to alleviate fructose-induced metabolic impairments. Thus, nineteen Wistar rats fed on a basal commercial feed were supplemented with either 30% (w/v) fructose in drinking water (F; n = 7) or 30% (w/v) fructose solution plus 0.2% (w/v) P (F + P; n = 6) for 10 weeks. The results were compared to those from a control group fed on the basal diet and provided with drinking water (C; n = 6). Body weight and energy intake were registered weekly. P supplementation decreased fat depots, counteracted the dyslipidemia caused by F and improved markers of liver injury including steatosis. The study of the microbiota by metagenomics and urine by untargeted MS-based metabolomics revealed microbial metabolites from P that may be responsible for these health benefits.

The pro-oxidant action of high-oxygen MAP on beef patties can be counterbalanced by antioxidant compounds from common hawthorn and rose hips.

The objective of this research was to evaluate the effectiveness of antioxidant-rich extracts from rose hip (Rosa canina L.; RC) and hawthorn (Crataegus monogyna Jacq.; CM) at minimizing the oxidative damage to proteins and lipids in beef patties subjected to a high­oxygen (HiOx-MAP) and vacuum (Vacuum) packaging atmosphere. The extracts of RC and CM were characterized by quantifying bioactive compounds, namely, phenolic compounds, tocopherols and vitamin C. Both fruits had high concentrations of bioactive compounds, with RC having the highest total phenolic and vitamin C content. Yet, CM was the most efficient in protecting beef patties against protein carbonylation, reducing, as a result, the instrumental toughness in cooked beef patties. The use of CM and RC extracts in beef patties significantly improved consumer purchase intention in HiOx-MAP packaging systems. The use of CM and RC extracts or their combination in future research would be an effective antioxidant means to decrease the pro-oxidative effects caused by HiOx-MAP in red meat.

Biodegradation of Punicalagin into Ellagic Acid by Selected Probiotic Bacteria: A Study of the Underlying Mechanisms by MS-Based Proteomics.

Pomegranate (Punica granatum L.) is a well-known source of bioactive phenolic compounds such as ellagitannins, anthocyanins, and flavanols. Punicalagin, one of the main constituents of pomegranate, needs to be biodegraded by bacteria to yield metabolites of medicinal interest. In this work, we tested 30 lactic acid bacteria (LAB) and their capacity to transform punicalagin from a punicalagin-rich pomegranate extract into smaller bioactive molecules, namely, ellagic acid and urolithins. These were identified and quantified by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS2). Further, we evaluated the molecular mechanism governing this transformation through label-free comparative MS-based proteomics. All tested LAB strains were capable of transforming punicalagin into ellagic acid, while the biosynthesis of urolithins was not observed. Proteomic analysis revealed an increase of generic transglycosylases that might have a hydrolytic role in the target phenolic molecule, coupled with an increase in the quantity of ATP-binding cassette (ABC) transporters, which might play a relevant role in transporting the resulting byproducts in and out of the cell.

The D165H Polymorphism of QiMYB-like-1 Is Linked to Interactions between Tannin Accumulation, Herbivory and Biogeographical Determinants of Quercus ilex.

The accumulation in the leaves and young stems of phenolic compounds, such as hydrolyzable and condensed tannins, constitutes a defense mechanism of plants against herbivores. Among other stressing factors, chronic herbivory endangers Quercus ilex, a tree playing a central role in Mediterranean forests. This work addressed the connections between the chemical defenses of Q. ilex leaves and their susceptibility to herbivory, quantitative traits whose relationships are modulated by environmental and genetic factors that could be useful as molecular markers for the selection of plants with improved fitness. A search for natural variants detected the polymorphism D165H in the effector domain of QiMYB-like-1, a TT2-like transcription factor whose family includes members that control the late steps of condensed tannins biosynthesis in different plant species. QiMYB-like-1 D165H polymorphism was screened by PCR-RFLP in trees from six national parks in Spain where Q. ilex has a relevant presence, revealing that, unlike most regions that match the Hardy-Weinberg equilibrium, homozygous plants are over-represented in "Monfragüe" and "Cabañeros", among the best examples to represent the continental Mediterranean (cM) ecosystem. Accordingly, the averages of two stress-related quantitative traits measured in leaves, herbivory index and accumulation of condensed tannins, showed asymmetric distributions depending on the clustering of trees based on ecological and genetic factors. Thus, the impact of herbivory was greater in managed forests with a low density of trees from the cM region, among which QiMYB-like-1 D165 homozygotes stand out, whereas condensed tannins accumulation was higher in leaves of QiMYB-like-1 H165 homozygotes from low-density forests, mainly in the Pyrenean (Py) region. Besides, the correlation between the contents of condensed tannins and total tannins vanished after clustering by the same factors: the cM region singularity, forest tree density, and QiMYB-like-1 genotype, among which homozygous shared the lowest link. The biogeographical and genetic constraints that modulate the contribution of condensed tannins to chemical defenses also mediated their interactions with the herbivory index, which was found positively correlated with total phenolics or tannins, suggesting an induction signal by this biotic stress. In contrast, a negative correlation was observed with condensed tannins after tree clustering by genetics factors where associations between tannins were lost. Therefore, condensed tannins might protect Q. ilex from defoliation in parks belonging to the cM ecosystem and carrying genetic factor(s) linked to the QiMYB-like-1 D165H polymorphism.

Hormone and secondary metabolite profiling in chestnut during susceptible and resistant interactions with Phytophthora cinnamomi.

Phytophthora cinnamomi (Pc) is a dangerous pathogen that causes root rot (ink disease) and threatens the production of chestnuts worldwide. Despite all the advances recently reported at molecular and physiological level, there are still gaps of knowledge that would help to unveil the defence mechanisms behind plant-Pc interactions. Bearing this in mind we quantified constitutive and Pc-induced stress-related signals (hormones and metabolites) complemented with changes in photosynthetic related parameters by exploring susceptible and resistant Castanea spp.-Pc interactions. In a greenhouse experiment, five days before and nine days after inoculation with Pc, leaves and fine roots from susceptible C. sativa and resistant C. sativa × C. crenata clonal 2-year-old plantlets were sampled (clones Cs14 and 111-1, respectively). In the resistant clone, stomatal conductance (gs) and net photosynthesis (A) decreased significantly and soluble sugars in leaves increased, while in the susceptible clone gs and A remained unchanged and proline levels in leaves increased. In the resistant clone, higher constitutive content of root SA and foliar ABA, JA and JA-Ile as compared to the susceptible clone were observed. Total phenolics and condensed tannins were highest in roots of the susceptible clone. In response to infection, a dynamic hormonal response in the resistant clone was observed, consisting of accumulation of JA, JA-Ile and ABA in roots and depletion of total phenolics in leaves. However, in the susceptible clone only JA diminished in leaves and increased in roots. Constitutive and Pc-induced levels of JA-Ile were only detectable in the resistant clone. From the hormonal profiles obtained in leaves and roots before and after infection, it is concluded that the lack of effective hormonal changes in C. sativa explains the lack of defence responses to Pc of this susceptible species.

Regulation by biotic stress of tannins biosynthesis in Quercus ilex: Crosstalk between defoliation and Phytophthora cinnamomi infection.

Sustainability of the Mediterranean forest is threatened by oak decline, a disease of holm oak and other Quercus species that is initiated by infection with the oomycete Phytophthora cinnamomi. Focusing on the role of tannins in the chemical defense of plants, this work investigated whether tannins content in Quercus ilex is regulated by biotic stress. Screening of published genomes allowed the identification of Quercus sequences encoding enzymes for early steps of the biosynthesis of phenolic compounds, like hydrolysable tannins and condensed tannins (CT) among others, plus genes involved in the late steps of CT biosynthesis. Four days after treatment of Q. ilex seedlings by mechanical defoliation, P. cinnamomi infection and both stressors simultaneously, mRNA concentrations for tannins biosynthesis enzymes were measured in leaves. Among the transcript amount for shikimate dehydrogenase (SDH, EC 1.1.1.25), anthocyanidin reductase (EC 1.3.1.77), anthocyanidin synthase (EC 1.14.11.19) and leucoanthocyanidine reductase (EC 1.17.1.3), defoliation induced gene expression for SDH2 isoenzyme. About 4 days after infection of roots by P. cinnamomi, this up-regulation was canceled and SDH enzyme activity decreased. Furthermore, during this late stage of biotrophic interaction the pathogen switched off the correlation engaged by defoliation between the expression of SDH1 and SDH2 encoding genes and chemical defenses corresponding to total tannins, which were down-regulated. Thus, tannins biosynthesis in seedlings of Q. ilex is induced after mechanical defoliation whereas infection by the pathogen interferes with this regulation, potentially increasing the susceptibility of plants to herbivory and aggravating the impact of biotic stress.

Prolonging shelf life of lamb cutlets packed under high-oxygen modified atmosphere by spraying essential oils from North-African plants.

The aim of this study was to assess the effectiveness of sprayable essential oils for counteracting the pro-oxidant environment created during retail display of lamb cutlets packed under high-oxygen atmosphere. North-African essential oils from Pituranthos chloranthus (PC) and Teucrium ramosissimum (TR) were applied by spraying on the surface of whole-lamb cutlets. The bio-preservative treatment led to a significant increase of their shelf life in terms of protection against lipid and protein oxidative deterioration, discoloration of lean and bone and microbiological stability during 12days of chilled storage. Furthermore, the addition of TR and PC on the surface of lamb cutlets significantly improved their appearance, increasing hence the purchase intention and consumers' preferences towards treated retailed products. This study shows that the application of a high-oxygen active packaging in combination with natural sprayable extracts is a feasible and effective strategy for protecting whole-meat muscle foods against oxidation and microbial deterioration.

Seasonal changes in the sperm fatty acid composition of Shetland pony stallions.

Spermatozoa contain polyunsaturated fatty acids (PUFA). Cryopreservation damages sperm membranes and they become less functional after thawing. We analysed the lipid composition of spermatozoa from Shetland stallions (n = 15) collected monthly from January to June and hypothesized that sperm lipid patterns change with season. In addition, one ejaculate per month was submitted to cryopreservation. Content of saturated palmytic and stearic acid decreased from January to March (p < 0.001) while content of the PUFA docosapentaenoic (p < 0.001) and arachidonic acid (p < 0.05) and total PUFA (p < 0.001) increased. Docosapentaenoic acid was the predominant PUFA in stallion spermatozoa. In conclusion, the sperm fatty acid composition of Shetland pony stallions undergoes seasonal changes, and PUFA content increases from the non-breeding to the breeding season. Seasonal differences in sperm fatty acids might in part explain seasonal differences in the resistance of equine spermatozoa to cryopreservation and cooled-storage.

Antioxidant Extracts from Acorns (Quercus ilex L.) Effectively Protect Ready-to-Eat (RTE) Chicken Patties Irrespective of Packaging Atmosphere.

This study evaluated the impact of a phenolic-rich acorn extract (200 ppm gallic acid equivalents) and the concentration of oxygen in the packaging system (low-oxygen modified atmosphere; 5% vs. normal-oxygen; 21%) on lipid and protein oxidation and consumers acceptance of the ready-to-eat chicken patties. Samples were subjected to cooking (electric oven, 170 °C/16 min), cold storage (14 d at 4 °C), and reheating (microwave, 600 mW/1 min). Samples treated with acorn extract kept thiobarbituric acid-reactive substances numbers and lipid-derived volatiles at basal levels throughout the whole processing irrespective of the oxygen concentration in the packaging atmosphere. Consistently, treated patties had lower protein carbonyls than control ones. The acorn extract also controlled color and texture deterioration during chilled storage and reheating and improved the color and odor acceptance of the products. Formulating with acorn extract is a feasible strategy to inhibit the oxidation-driven changes and preserve the quality of reheated samples as if there were freshly cooked. Compared to the effect of the antioxidant extract, the concentration of oxygen in the packaging system was negligible in terms of quality preservation.

Effect of pre-cooking methods on the chemical and sensory deterioration of ready-to-eat chicken patties during chilled storage and microwave reheating.

The effects of pre-cooking methods, namely, boiling (BL), roasting (RT) and grilling (GR), refrigerated storage (14 days/+4 °C) and microwave reheating on chicken patties were studied. Physical, chemical and sensory parameters were evaluated in order to correlate the chemical deterioration of ready-to-eat chicken patties with the acceptance of the odor. Chemical deterioration was evaluated through the chemical composition, Maillard compounds, Thiobarbituric acid-reactive substances (TBARS) and volatiles. Sensory deterioration (odor liking) was performed by an acceptance test with hedonic scale. According to the TBARS values and volatile compounds generated in the head space during the examined stages, the pre-cooking method and the storage time had a significant effect on lipid oxidation, whereas reheating in a microwave had a negligible impact. At each succeeding processing stage, panelists gave lower odor scores to all samples and no significant differences were found between treatments at any stage. RT and GR patties showed less intense chemical changes and presented higher acceptation scores by the sensory panel than BL patties. Thus, the choice of pre-cooking method and control of storage conditions plays a key role in the inhibition of oxidative changes in ready-to-eat chicken patties.

Apple phenolics as inhibitors of the carbonylation pathway during in vitro metal-catalyzed oxidation of myofibrillar proteins.

The effect of apple phenolics on the oxidative damage caused to myofibrillar proteins by an in vitro metal-catalyzed oxidation system was investigated. Three pure phenolic compounds (chlorogenic acid, (-)-epicatechin and phloridzin) and an apple peel extract were added to myofibrillar proteins in three concentrations (50, 100 and 200µM), and a blank treatment was included as a control. All suspensions were subjected to Fe(3+)/H2O2 oxidation at 37°C during 10days, and protein oxidation was evaluated as carbonylation (α-amino adipic and γ-glutamic semialdehydes) and Schiff base cross-links. Significant inhibition by apple phenolics was found as compared to the control treatment, with (-)-epicatechin being the most efficient antioxidant and phloridzin showing the weakest antioxidant effect. The higher concentrations of apple extract showed effective antioxidant activity against protein oxidation in myofibrillar proteins, emphasizing the potential of apple by-products as natural inhibitors of protein oxidation in meat products.

The application of natural antioxidants via brine injection protects Iberian cooked hams against lipid and protein oxidation.

In response to the increasing consumers' mistrust in synthetic additives, the meat industry is focused on searching sources of natural antioxidants. Two different sources of natural antioxidants i) a mixture of garlic, cinnamon, cloves and rosemary essential oils and ii) a Rosa canina L. extract, were compared with a commercial antioxidant additive (Artinox®) for their ability to control protein and lipid oxidation in cooked hams after a settling period of 30 days and at the end of a chilled storage (150 days). The mixture of essential oils was the most effective against lipid oxidation while R. canina L. extracts were the most effective in controlling protein carbonylation at day 150. Accordingly, the use of these antioxidants via brine injection is a successful strategy to enhance the oxidative stability of cooked hams without modifying their physicochemical properties.

Influence of the Oxidation States of 4-Methylcatechol and Catechin on the Oxidative Stability of ß-Lactoglobulin.

Chemical interactions between proteins and phenols affect the overall oxidative stability of a given biological system. To investigate the effect of protein-phenol adduct formation on the oxidative stability of ß-lactoglobulin (ß-LG), the protein was left to react with an equimolar concentration of 4-methylcatechol (4MC), catechin (Cat), or their respective quinone forms, 4-methylbenzoquinone (4MBQ) and catechin-quinone (CatQ), and subsequently subjected to metal-catalyzed oxidation by Fe(II)/H2O2 for 20 days at 37 °C. The reaction with 4MBQ resulted in 60% thiol loss and 22% loss of amino groups, whereas the addition of 4MC resulted in 12% thiol loss. The reaction with Cat or CatQ resulted in no apparent modification of ß-LG. The oxidative stability of ß-LG after reaction with each of 4MC, 4MBQ, Cat, or CatQ was impaired. Especially 4MC and 4MBQ were found to be pro-oxidative toward α-aminoadipic semialdehyde and γ-glutamic semialdehyde formation as well as the generation of fluorescent Schiff base products. The changes observed were ascribed to the redirection of oxidation as a result of the blocking of thiol groups but also to the oxidative deamination pathway, accelerating the production of semialdehydes and subsequently Schiff base structures.

Temperature of frozen storage affects the nature and consequences of protein oxidation in beef patties.

The effect of three frozen storage temperatures (-8, -18 and -80 °C) on protein oxidation in beef patties was studied through the analysis of novel oxidation markers. Additionally, the connection between lipid and protein oxidation and the impact of the latter on particular quality traits (water holding capacity, color and texture) of subsequently processed beef patties (cooking/cold-stored) were investigated. Protein oxidation was measured as the loss of tryptophan fluorescence and formation of diverse lysine oxidation products (α-aminoadipic semialdehyde, α-aminoadipic acid and Schiff bases). Lipid oxidation was assessed by levels of thiobarbituric acid reactive substances and hexanal. A significant effect of storage temperature on protein oxidation was detected. Frozen storage increased the susceptibility of meat proteins to undergo further oxidation during processing. Timely interactions were found between lipid and protein oxidation. Plausible mechanisms by which oxidative damage to proteins may have an impact in particular quality traits are thoroughly discussed.

Effect of intramuscular fat content and serving temperature on temporal sensory perception of sliced and vacuum packaged dry-cured ham.

The present study aimed to evaluate the influence of three serving temperatures (7 °C, 16 °C and 20 °C) and two different ham sections varying in the intramuscular fat (IMF) content on the sensory characteristics of sliced and vacuum-packaged Iberian dry-cured hams using the time-intensity (TI) method. Preceding the TI study, appearance and odour of dry-cured hams were evaluated using a descriptive profile. Fluidity and brightness of the external fat, brightness of lean and all odour attributes increased as serving temperature increased whereas the hardness of external fat decreased with temperature. Oral temperature would have disguised the effect of serving temperature over time as a consequence of a possible balance between both temperatures during samples' consumption. TI revealed that the effect of serving temperature on flavour and texture perception was more noticeable along the first seconds of chewing. Odour intensities increased with the IMF content and temporal perception of hardness, saltiness and rancid flavour were also significantly influenced by the IMF content.

Dog rose (Rosa canina L.) as a functional ingredient in porcine frankfurters without added sodium ascorbate and sodium nitrite.

The effect of dog rose (Rosa canina L.; RC), rich in polyphenols and ascorbic acid, on lipid and protein oxidation, colour stability and texture of frankfurters was investigated. Four treatments were prepared: with 5 or 30 g/kg RC extract and without sodium ascorbate and sodium nitrite (5RC and 30RC, respectively), a positive control (with sodium ascorbate and sodium nitrite; PC) and a negative control (without sodium ascorbate, sodium nitrite or RC extract; NC). Hexanal values were much higher throughout storage in NC compared to RC and PC frankfurters (P<0.001). The RC extracts protected against protein oxidation, but not as efficiently as PC (P<0.05). In the RC treated frankfurters, lower a* values were measured compared to PC due to the lack of sodium nitrite. In conclusion, dog rose can act as a natural antioxidant in frankfurters, but not as full replacer for sodium nitrite.

Formation of lysine-derived oxidation products and loss of tryptophan during processing of porcine patties with added avocado byproducts.

The effects of the addition of avocado oil and a phenolic-rich avocado peel extract on protein oxidation were studied in porcine patties subjected to cooking and chilled storage. Protein oxidation was assessed by means of tryptophan loss and the formation of specific lysine oxidation products, such as α-aminoadipic semialdehyde (AAS), α-aminoadipic acid (AAA), and Schiff bases. In the present paper, quantitative data of AAA are reported for the first time on a food matrix. The addition of the avocado extract inhibited the formation of AAS, AAA, and Schiff bases in patties during cooking and subsequent chilled storage. The antioxidant effect may respond to the protecting effect of phenolic compounds, mainly procyanidins, found on the avocado extract. Apparently, the combination of both strategies (back-fat replacement and addition of avocado extract) does not lead to an enhanced advantage on the oxidative stability of the product. The novel methodologies used in the present study enable a better comprehension of the mechanisms and consequences of protein oxidation in food systems.

Inhibition of cholesterol oxidation products (COPs) formation in emulsified porcine patties by phenolic-rich avocado (Persea americana Mill.) extracts.

The effect of phenolic-rich extracts from avocado peel on the formation of cholesterol oxidation products (COPs) in porcine patties subjected to cooking and chill storage was studied. Eight COPs (7α-hydroxycholesterol, 7ß-hydroxycholesterol, 7-ketocholesterol, 20α-hydroxycholesterol, 25-hydroxycholesterol, cholestanetriol, 5,6ß-epoxycholesterol, and 5,6α-epoxycholesterol) were identified and quantified by GC-MS. The addition of avocado extracts (∼600 GAE/kg patty) to patties significantly inhibited the formation of COPs during cooking. Cooked control (C) patties contained a larger variety and greater amounts of COPs than the avocado-treated (T) counterparts. COPs sharply increased in cooked patties during the subsequent chilled storage. This increase was significantly higher in C patties than in the T patties. Interestingly, the amount of COPs in cooked and chilled T patties was similar to those found in cooked C patties. The mechanisms implicated in cholesterol oxidation in a processed meat product, the protective effect of avocado phenolics, and the potential implication of lipid and protein oxidation are thoroughly described in the present paper.

Fluorescent HPLC for the detection of specific protein oxidation carbonyls - α-aminoadipic and γ-glutamic semialdehydes - in meat systems.

Precise methodologies for the routine analysis of particular protein carbonyls are required in order to progress in this topic of increasing interest. The present paper originally describes the application of an improved method for the detection of α-aminoadipic and γ-glutamic semialdehydes in a meat system by using a derivatization procedure with p-amino-benzoic acid (ABA) followed by fluorescent high-performance liquid chromatography (HPLC). The method development comprises i) the description of a simple HPLC program which allows the efficient separation of the ABA and the key standard compounds and ii) the optimization of the procedure for the preparation of a meat sample in order to maximize the fluorescent signal for both protein carbonyls. Furthermore, the suitability of this method is evaluated by applying the technique to porcine burger patties. The present procedure enables an accurate and relatively fast analysis of both semialdehydes in meat samples in which they could play an interesting role as reliable indicators of protein oxidation.

Avocado (Persea americana Mill.) phenolics, in vitro antioxidant and antimicrobial activities, and inhibition of lipid and protein oxidation in porcine patties.

The first aim of the present work (study 1) was to analyze ethyl acetate, 70% acetone, and 70% methanol extracts of the peel, pulp, and seed from two avocado (Persea americana Mill.) varieties, namely, 'Hass' and 'Fuerte', for their phenolic composition and their in vitro antioxidant activity using the CUPRAC, DPPH, and ABTS assays. Their antimicrobial potential was also studied. Peels and seeds had higher amounts of phenolics and a more intense in vitro antioxidant potential than the pulp. Peels and seeds were rich in catechins, procyanidins, and hydroxycinnamic acids, whereas the pulp was particularly rich in hydroxybenzoic and hydroxycinnamic acids and procyanidins. The total phenolic content and antioxidant potential of avocado phenolics was affected by the extracting solvent and avocado variety. The avocado materials also displayed moderate antimicrobial effects against Gram-positive bacteria. Taking a step forward (study 2), extracts (70% acetone) from avocado peels and seeds were tested as inhibitors of oxidative reactions in meat patties. Avocado extracts protected meat lipids and proteins against oxidation with the effect on lipids being dependent on the avocado variety.