Cláudia N. Santos

A standardised static in vitro digestion method suitable for food-an international consensus

Simulated gastro-intestinal digestion is widely employed in many fields of food and nutritional sciences, as conducting human trials are often costly, resource intensive, and ethically disputable. As a consequence, in vitro alternatives that determine endpoints such as the bioaccessibility of nutrients and non-nutrients or the digestibility of macronutrients (e.g. lipids, proteins and carbohydrates) are used for screening and building new hypotheses. Various digestion models have been proposed, often impeding the possibility to compare results across research teams. For example, a large variety of enzymes from different sources such as of porcine, rabbit or human origin have been used, differing in their activity and characterization. Differences in pH, mineral type, ionic strength and digestion time, which alter enzyme activity and other phenomena, may also considerably alter results. Other parameters such as the presence of phospholipids, individual enzymes such as gastric lipase and digestive emulsifiers vs. their mixtures (e.g. pancreatin and bile salts), and the ratio of food bolus to digestive fluids, have also been discussed at length. In the present consensus paper, within the COST Infogest network, we propose a general standardised and practical static digestion method based on physiologically relevant conditions that can be applied for various endpoints, which may be amended to accommodate further specific requirements. A frameset of parameters including the oral, gastric and small intestinal digestion are outlined and their relevance discussed in relation to available in vivo data and enzymes. This consensus paper will give a detailed protocol and a line-by-line, guidance, recommendations and justifications but also limitation of the proposed model. This harmonised static, in vitro digestion method for food should aid the production of more comparable data in the future.

The role of plant defence proteins in fungal pathogenesis.

SUMMARY It is becoming increasingly evident that a plant-pathogen interaction may be compared to an open warfare, whose major weapons are proteins synthesized by both organisms. These weapons were gradually developed in what must have been a multimillion-year evolutionary game of ping-pong. The outcome of each battle results in the establishment of resistance or pathogenesis. The plethora of resistance mechanisms exhibited by plants may be grouped into constitutive and inducible, and range from morphological to structural and chemical defences. Most of these mechanisms are defensive, exhibiting a passive role, but some are highly active against pathogens, using as major targets the fungal cell wall, the plasma membrane or intracellular targets. A considerable overlap exists between pathogenesis-related (PR) proteins and antifungal proteins. However, many of the now considered 17 families of PR proteins do not present any known role as antipathogen activity, whereas among the 13 classes of antifungal proteins, most are not PR proteins. Discovery of novel antifungal proteins and peptides continues at a rapid pace. In their long coevolution with plants, phytopathogens have evolved ways to avoid or circumvent the plant defence weaponry. These include protection of fungal structures from plant defence reactions, inhibition of elicitor-induced plant defence responses and suppression of plant defences. A detailed understanding of the molecular events that take place during a plant-pathogen interaction is an essential goal for disease control in the future.

In Vitro Models for Studying Secondary Plant Metabolite Digestion and Bioaccessibility

There is an increased interest in secondary plant metabolites, such as polyphenols and carotenoids, due to their proposed health benefits. Much attention has focused on their bioavailability, a prerequisite for further physiological functions. As human studies are time consuming, costly, and restricted by ethical concerns, in vitro models for investigating the effects of digestion on these compounds have been developed and employed to predict their release from the food matrix, bioaccessibility, and assess changes in their profiles prior to absorption. Most typically, models simulate digestion in the oral cavity, the stomach, the small intestine, and, occasionally, the large intestine. A plethora of models have been reported, the choice mostly driven by the type of phytochemical studied, whether the purpose is screening or studying under close physiological conditions, and the availability of the model systems. Unfortunately, the diversity of model conditions has hampered the ability to compare results across different studies. For example, there is substantial variability in the time of digestion, concentrations of salts, enzymes, and bile acids used, pH, the inclusion of various digestion stages; and whether chosen conditions are static (with fixed concentrations of enzymes, bile salts, digesta, and so on) or dynamic (varying concentrations of these constituents). This review presents an overview of models that have been employed to study the digestion of both lipophilic and hydrophilic phytochemicals, comparing digestive conditions in vitro and in vivo and, finally, suggests a set of parameters for static models that resemble physiological conditions.

Mind the gap—deficits in our knowledge of aspects impacting the bioavailability of phytochemicals and their metabolites—a position paper focusing on carotenoids and polyphenols

Various secondary plant metabolites or phytochemicals, including polyphenols and carotenoids, have been associated with a variety of health benefits, such as reduced incidence of type 2 diabetes, cardiovascular diseases, and several types of cancer, most likely due to their involvement in ameliorating inflammation and oxidative stress. However, discrepancies exist between their putative effects when comparing observational and intervention studies, especially when using pure compounds. These discrepancies may in part be explained by differences in intake levels and their bioavailability. Prior to exerting their bioactivity, these compounds must be made bioavailable, and considerable differences may arise due to their matrix release, changes during digestion, uptake, metabolism, and biodistribution, even before considering dose‐ and host‐related factors. Though many insights have been gained on factors affecting secondary plant metabolite bioavailability, many gaps still exist in our knowledge. In this position paper, we highlight several major gaps in our understanding of phytochemical bioavailability, including effects of food processing, changes during digestion, involvement of cellular transporters in influx/efflux through the gastrointestinal epithelium, changes during colonic fermentation, and their phase I and phase II metabolism following absorption.

Phosphorylation modulates clearance of alpha-synuclein inclusions in a yeast model of Parkinson's disease.

Alpha-synuclein (aSyn) is the main component of proteinaceous inclusions known as Lewy bodies (LBs), the typical pathological hallmark of Parkinsons disease (PD) and other synucleinopathies. Although aSyn is phosphorylated at low levels under physiological conditions, it is estimated that ∼90% of aSyn in LBs is phosphorylated at S129 (pS129). Nevertheless, the significance of pS129 in the biology of aSyn and in PD pathogenesis is still controversial. Here, we harnessed the power of budding yeast in order to assess the implications of phosphorylation on aSyn cytotoxicity, aggregation and sub-cellular distribution. We found that aSyn is phosphorylated on S129 by endogenous kinases. Interestingly, phosphorylation reduced aSyn toxicity and the percentage of cells with cytosolic inclusions, in comparison to cells expressing mutant forms of aSyn (S129A or S129G) that mimic the unphosphorylated form of aSyn. Using high-resolution 4D imaging and fluorescence recovery after photobleaching (FRAP) in live cells, we compared the dynamics of WT and S129A mutant aSyn. While WT aSyn inclusions were very homogeneous, inclusions formed by S129A aSyn were larger and showed FRAP heterogeneity. Upon blockade of aSyn expression, cells were able to clear the inclusions formed by WT aSyn. However, this process was much slower for the inclusions formed by S129A aSyn. Interestingly, whereas the accumulation of WT aSyn led to a marked induction of autophagy, cells expressing the S129A mutant failed to activate this protein quality control pathway. The finding that the phosphorylation state of aSyn on S129 can alter the ability of cells to clear aSyn inclusions provides important insight into the role that this posttranslational modification may have in the pathogenesis of PD and other synucleinopathies, opening novel avenues for investigating the molecular basis of these disorders and for the development of therapeutic strategies.

Fungal Pathogens: The Battle for Plant Infection

The attempted infection of a plant by a pathogen, such as a fungus or an Oomycete, may be regarded as a battle whose major weapons are proteins and smaller chemical compounds produced by both organisms. Indeed, plants produce an astonishing plethora of defense compounds that are still being discovered at a rapid pace. This pattern arose from a multi-million year, ping-pong−type co-evolution, in which plant and pathogen successively added new chemical weapons in this perpetual battle. As each defensive innovation was established in the host, new ways to circumvent it evolved in the pathogen. This complex co-evolution process probably explains not only the exquisite specificity observed between many pathogens and their hosts, but also the ineffectiveness or redundancy of some defensive genes which often encode enzymes with overlapping activities. Plants evolved a complex, multi-level series of structural and chemical barriers that are both constitutive or preformed and inducible. These defenses may involve strengthening of the cell wall, hypersensitive response (HR), oxidative burst, phytoalexins and pathogenesis-related (PR) proteins. The pathogen must successfully overcome these obstacles before it succeeds in causing disease. In some cases, it needs to modulate or modify plant cell metabolism to its own benefit and/or to abolish defense reactions. Central to the activation of plant responses is timely perception of the pathogen by the plant. A crucial role is played by elicitors which, depending on their mode of action, are broadly classified into nonspecific elicitors and highly specific elicitors or virulence effector/avirulence factors. A protein battle for penetration is then initiated, marking the pathogen attempted transition from extracellular to invasive growth before parasitism and disease can be established. Three major types of defense responses may be observed in plants: non-host resistance, host resistance, and host pathogenesis. Plant innate immunity may comprise a continuum from non-host resistance involving the detection of general elicitors to host-specific resistance involving detection of specific elicitors by R proteins. It was generally assumed that non-host resistance was based on passive mechanisms and that nonspecific rejection usually arose as a consequence of the non-host pathogen failure to breach the first lines of plant defense. However, recent evidence has blurred the clear-cut distinction among non-host resistance, host-specific resistance and disease. The same obstacles are also serious challenges for host pathogens, reducing their success rate significantly in causing disease. Indeed, even susceptible plants mount a (insufficient) defense response upon recognition of pathogen elicited molecular signals. Recent evidence suggests the occurrence of significant overlaps between the protein components and signalling pathways of these types of resistance, suggesting the existence of both shared and unique features for the three branches of plant innate immunity.

Antioxidant Properties and Neuroprotective Capacity of Strawberry Tree Fruit (Arbutus unedo)

Berries contain significant amounts of phytochemicals, including polyphenols, which are reported to reduce cancer risk, coronary heart disease and other degenerative diseases. These effects are mainly attributed to the antioxidant capacity of polyphenols found in berries. Strawberry tree (Arbutus unedo) berries are used in folk medicine but seldom eaten as fresh fruits. Their phenolic profile and antioxidant capacity reveal a high potential, but they are not well characterized as a “health promoting food”. The aim of this study was to assess the antioxidant properties of the edible strawberry tree fruit in vitro and in a neurodegeneration cell model. Raspberry (Rubus idaeus), a well documented health-promoting fruit, was used as a control for comparison purposes. A. unedo yielded a similar content in polyphenols and a slightly lower value of total antioxidant capacity in comparison to R. idaeus. Although the chemically-measured antioxidant activity was similar between both fruits, R. idaeus increased neuroblastoma survival in a neurodegeneration cell model by 36.6% whereas A. unedo extracts caused no effect on neuroblastoma viability. These results clearly demonstrate that a promising level of chemically-determined antioxidant activity of a plant extract is not necessarily correlated with biological significance, as assessed by the effect of A. unedo fruit in a neurodegeneration cell model.

Antioxidant capacity of Macaronesian traditional medicinal plants.

The use of many traditional medicinal plants is often hampered by the absence of a proper biochemical characterization, essential to identify the bioactive compounds present. The leaves from five species endemic to the Macaronesian islands with recognized ethnobotanical applications were analysed: Apollonias barbujana (Cav.) Bornm., Ocotea foetens (Ainton) Baill, Prunus azorica (Mouill.) Rivas-Mart., Lousã, Fern. Prieto, E. Días, J.C. Costa & C. Aguiar, Rumex maderensis Lowe and Plantago arborescens Poir. subsp. maderensis (Dcne.) A. Hans. et Kunk.. Since oxidative stress is a common feature of most diseases traditionally treated by these plants, it is important to assess their antioxidant capacity and determine the molecules responsible for this capacity. In this study, the antioxidant capacity of these plants against two of the most important reactive species in human body (hydroxyl and peroxyl radicals) was determined. To trace the antioxidant origin total phenol and flavonoid contents as well as the polyphenolic profile and the amount of trace elements were determined. There was a wide variation among the species analysed in what concerns their total leaf phenol and flavonoid contents. From the High Performance Liquid Chromatography (HPLC) electrochemically detected peaks it was possible to attribute to flavonoids the antioxidant capacity detected in A. barbujana, O. foetens, R. maderensis and P. azorica extracts. These potential reactive flavonoids were identified for A. barbujana, R. maderensis and P. azorica. For R. maderensis a high content (7 mg g-1 dry weight) of L-ascorbic acid, an already described antioxidant phytomolecule, was found. A high content in selenomethionine (414.35 μg g-1 dry weight) was obtained for P. arborescens subsp. maderensis extract. This selenocompound is already described as a hydroxyl radical scavenger is reported in this work as also possessing peroxyl radical scavenging capacity. This work is a good illustration of different phytomolecules (flavonoids, organic acids and selenocompounds), presents in leaves of the five traditional medicinal plants endemic to Macaronesia, all exhibiting antioxidant properties.

Neuroprotective effects of digested polyphenols from wild blackberry species

PurposeBlackberry ingestion has been demonstrated to attenuate brain degenerative processes with the benefits ascribed to the (poly)phenolic components. The aim of this work was to evaluate the neuroprotective potential of two wild blackberry species in a neurodegeneration cell model and compare them with a commercial variety.MethodsThis work encompasses chemical characterization before and after an in vitro digestion and the assessment of neuroprotection by digested metabolites. Some studies targeting redox/cell death systems were also performed to assess possible neuroprotective molecular mechanisms.ResultsThe three blackberry extracts presented some quantitative differences in polyphenol composition that could be responsible for the different responses in the neurodegeneration cell model. Commercial blackberry extracts were ineffective but both wild blackberries, Rubus brigantinus and Rubus vagabundus, presented neuroprotective effects. It was verified that a diminishment of intracellular ROS levels, modulation of glutathione levels and activation of caspases occurred during treatment. The last effect suggests a preconditioning effect since caspase activation was not accompanied by diminution in cell death and loss of functionality.ConclusionsThis is the first time that metabolites obtained from an in vitro digested food matrix, and tested at levels approaching the concentrations found in human plasma, have been described as inducing an adaptative response.

Contribution of Yap1 towards Saccharomyces cerevisiae adaptation to arsenic-mediated oxidative stress

In the budding yeast Saccharomyces cerevisiae, arsenic detoxification involves the activation of Yap8, a member of the Yap (yeast AP-1-like) family of transcription factors, which in turn regulates ACR2 and ACR3, genes encoding an arsenate reductase and a plasma-membrane arsenite-efflux protein respectively. In addition, Yap1 is involved in the arsenic adaptation process through regulation of the expression of the vacuolar pump encoded by YCF1 (yeast cadmium factor 1 gene) and also contributing to the regulation of ACR genes. Here we show that Yap1 is also involved in the removal of ROS (reactive oxygen species) generated by arsenic compounds. Data on lipid peroxidation and intracellular oxidation indicate that deletion of YAP1 and YAP8 triggers cellular oxidation mediated by inorganic arsenic. In spite of the increased amounts of As(III) absorbed by the yap8 mutant, the enhanced transcriptional activation of the antioxidant genes such as GSH1 (gamma- glutamylcysteine synthetase gene), SOD1 (superoxide dismutase 1 gene) and TRX2 (thioredoxin 2 gene) may prevent protein oxidation. In contrast, the yap1 mutant exhibits high contents of protein carbonyl groups and the GSSG/GSH ratio is severely disturbed on exposure to arsenic compounds in these cells. These results point to an additional level of Yap1 contribution to arsenic stress responses by preventing oxidative damage in cells exposed to these compounds. Transcriptional profiling revealed that genes of the functional categories related to sulphur and methionine metabolism and to the maintenance of cell redox homoeostasis are activated to mediate adaptation of the wild-type strain to 2 mM arsenate treatment.