Novel bioassays based on 3D-printed device for sensing of hypoxia and p53 pathway in 3D cell models.

Cell-based assays are widely exploited for drug screening and biosensing, providing useful information about bioactivity of target analytes and complex biological samples. It is well recognized that 3D cell models are required to achieve highly valuable information, also from the perspective of replacing animal models. However, bioassays relying on 3D cell models are generally highly demanding in terms of facilities, equipment, and skilled personnel requirements. To reduce cost, increase sustainability, and provide a flexible 3D cell-based platform for bioassays, we here report a novel approach based on a 3D-printed microtissue device. To assess the suitability of this strategy for reporter gene technology, we selected to monitor two molecular pathways which were of interest in several applications, hypoxia signaling and the p53 pathway. The investigation of such pathways is highly relevant in fields spanning from drug screening to bioactivity monitoring for industrial by-product valorization. Microtissues of human hepatocarcinoma (HepG2) and human embryonic kidney (Hek293T) cell lines were obtained with a low-cost and sustainable chip platform and bioassays were developed to monitor the hypoxia-inducible factors (HIFs) and the p53 tumor suppressor pathway. HepG2 and Hek293T 3D cell models were genetically engineered to express the Luc2P from Photinus pyralis firefly either under the regulation of p53 or HIF response elements. The bioassays allowed quantitative assessment of hypoxia and tumoral activity with 1,10-phenanthroline for HIF and with doxorubicin for p53 pathway activation, respectively, showing good potential for applications of this sustainable and low-cost 3D-printed microfluidic platform for bioactivity analyses, drug screening, and precision medicine.

New Insights on Primary and Secondary Metabolite Contents of Seven Italian Wild Food Plants with Medicinal Applications: A Comparative Study.

Wild food plants are widely consumed all over the world and many have both nutritional and therapeutic value due to the presence of biologically active compounds. The present research, for the first time, aims to compare primary and secondary metabolite levels among different plant organs (flower, leaf, stem, root, bark) of seven species (Borago officinalis L., Cynodon dactylon (L.) Pers., Foeniculum vulgare Mill., Hypericum perforatum L., Malva sylvestris L., Sambucus nigra L., Urtica dioica L.) collected in three different Italian regions (Liguria, Tuscany, Apulia). Plant organ samples were extracted with water or 95% (v/v) methanol and liquid fractions were analyzed using spectrophotometric assays. The best results were obtained for Hypericum perforatum L. samples, followed by Sambucus nigra L. and Borago officinalis L. As also confirmed via PCA analysis on normalized data, flower and leaf extracts of all species exhibited higher levels of polyphenols (up to 105.7 mg GA eq/gDW), reducing sugars (up to 389.2 mg GLUC eq/gDW), proteins (up to 675.7 mg BSA eq/gDW) and of antioxidant capacity (up to 263.5 mg AA eq/gDW). No differences among the regions of gathering were detected after spectrophotometric assays, which was confirmed via PCA analysis. These data contribute to further validate the traditionally reported healing effects of these species on human health.

Antioxidant and Sensory Properties of Raw and Cooked Pork Meat Burgers Formulated with Extract from Non-Compliant Green Coffee Beans.

The effects of polyphenol-rich extract obtained from non-compliant defatted green coffee beans (dGCBs) on physicochemical and antioxidant properties, as well as on the sensory profile of vacuum-packed pork burgers stored at 4 °C for 14 days and after cooking were assessed. The dGCB extract obtained by means of supercritical water extraction was analyzed for its polyphenol profile, total phenolic content, radical scavenging, and ferric-reducing antioxidant activities (DPPH and FRAP), Fe2+-chelating capacity, and total iron. The most abundant polyphenol component observed in the dGCB extract was chlorogenic acid, and the alkaloid caffeine was also present. This extract showed antioxidant properties. Thereafter, five formulations of pork meat burgers with added NaCl (1%) were prepared; one without the antioxidant (negative control, C) and one with the use of a synthetic antioxidant (0.05% ascorbic acid = positive control, A), while the other three were supplemented with a different amount of dGCB extract (P15 = 0.15%; P30 = 0.30%; P60 = 0.60%). The addition of dGCB extract increased the antioxidant activity of the raw and cooked burgers and reduced the lipid oxidation of the cooked burgers (0.47, 0.21, and 0.20 vs. 1.28 and 0.55 mg MDA eq./Kg, for P15, P30, and P60 vs. C and A, respectively). No negative effects were observed on the meat's color parameters and its stability during refrigerated storage and after cooking, nor on sensory attributes (color and aroma) for the lowest concentration of coffee extract. The results obtained indicate that 0.15% dGCB extract is a promising alternative to commercial synthetic antioxidants to improve the quality of refrigerated pork burgers.

Phytochemicals Recovery from Grape Pomace: Extraction Improvement and Chemometric Study.

In the last 20 years, an increased interest has been shown in the application of different types and combinations of enzymes to obtain phenolic extracts from grape pomace in order to maximize its valorization. Within this framework, the present study aims at improving the recovery of phenolic compounds from Merlot and Garganega pomace and at contributing to the scientific background of enzyme-assisted extraction. Five commercial cellulolytic enzymes were tested in different conditions. Phenolic compound extraction yields were analyzed via a Design of Experiments (DoE) methodology and a second extraction step with acetone was sequentially added. According to DoE, 2% w/w enzyme/substrate ratio was more effective than 1%, allowing a higher total phenol recovery, while the effect of incubation time (2 or 4 h) variation was more enzyme-dependent. Extracts were characterized via spectrophotometric and HPLC-DAD analyses. The results proved that enzymatic and acetone Merlot and Garganega pomace extracts were complex mixtures of compounds. The use of different cellulolytic enzymes led to different extract compositions, as demonstrated using PCA models. The enzyme effects were observed both in water enzymatic and in the subsequent acetone extracts, probably due to their specific grape cell wall degradation and leading to the recovery of different molecule arrays.

Ethnobotanical Review and Dataset Compiling on Wild and Cultivated Plants Traditionally Used as Medicinal Remedies in Italy.

Over the centuries, wild plants have constituted the main food ingredients and traditional medicine in rural communities. In the last decades, thousands of ethnobotanical studies have been conducted, with the aim of documenting the traditional knowledge on wild and cultivated plants both for food and therapeutic purposes. In the present work, 75 published papers related to Italian ethnobotanical knowledge on wild and cultivated plants traditionally used for medical purposes were analyzed and data on 1117 different species organized in the first dataset to target medicinal applications only. For each plant species, the Italian region of use, plant organs, mode of preparation, specific pathological group of application, citation index, and use index were listed. The different therapeutic applications were subdivided into nine main pathological groups according to the targeted human apparatus. Overall, the cited species with highest number of uses were related to the treatment of the digestive system and skin-ears-eyes-hair diseases, followed by diseases of the genito-urinary and respiratory systems. The 13 most relevant species were identified on the basis of their citation and use indexes. The present review on Italian medicinal flora aims to provide valuable information on wild and cultivated species, which are potential sources of plant-based therapeutic remedies, to preserve and reevaluate endangered traditional folk knowledge.

Looking for peptides from rice starch processing by-product: Bioreactor production, anti-tyrosinase and anti-inflammatory activity, and in silico putative taste assessment.

One of the major challenges for the modern society, is the development of a sustainable economy also aiming at the valorization of agro-industrial by-products in conjunction with at a significant reduction of generated residues from farm to retail. In this context, the present study demonstrates a biotechnological approach to yield bioactive peptides from a protein fraction obtained as a by-product of the rice starch production. Enzymatic hydrolysis, with the commercial proteases Alcalase and Protamex, were optimized in bioreactor up to 2 L of volume. The two best digestates, selected with respect to peptide release and extract antioxidant capacity, were further fractionated (cut-offs of 10, 5, and 1 kDa) via cross-flow filtration. Amino acid composition indicated that most of the fractions showed positive nutritional characteristics, but a putative bitter taste. A fraction obtained with Alcalase enzyme (retentate 8 kDa) exerted anti-inflammatory potential, while the smaller molecular weight fractions (retentate 1-5 kDa and permeate < 1 kDa) were more active in tyrosinase inhibition. The latter were further sub-fractionated by size-exclusion chromatography. From the 15 most anti-tyrosinase sub-fractions, 365 peptide sequences were identified via liquid chromatography coupled with high resolution mass spectrometry. The present data support the possible exploitation of bioactive peptide from rice starch by-product as ingredients into food, nutraceutical, pharmaceutical, and cosmetic formulations.

Extraction and Chemical Characterization of Functional Phenols and Proteins from Coffee (Coffea arabica) By-Products.

Not all the coffee produced goes to the roasting stage, because non-compliant green coffee beans are usually discarded by roasters and the silverskin of the coffee is usually removed and discarded. In the present work, non-compliant green coffee beans and coffee silverskins were fully characterized from a chemical point of view. In addition, enzyme-assisted extraction was applied to recover a fraction rich in proteins and polyphenols, tested for antimicrobial, antityrosinase, and antioxidant activities. Non-compliant green coffee beans showed higher amounts of polyphenols, flavanols, flavonoids, and caffeine than coffee silverskins (which were richer in tannins). The enzymatic extraction of non-compliant coffee green beans produced extracts with a good protein content and with a consistent quantity of polyphenols. The extract showed antioxidant, antityrosinase, and antimicrobial activity, thus representing a promising strategy to recover defective green coffee beans. The antioxidant and antimicrobial activity of coffee silver skins is lower than that of non-compliant coffee green beans extracts, while the antityrosinase activity is comparable.

Phytochemical characterization of raw and cooked traditionally consumed alimurgic plants.

In the past, wild edible alimurgic plants became an important alternative food source when poverty, wars or drought made it difficult to access crops. These plants were considered rich in highly nutritional compounds and also frequently used as food-medicine given their health-promoting properties. With the aim of improving our knowledge on the content of beneficial or detrimental compounds in relation with past local dietary and curative traditions, 12 wild food plant species were collected from two study areas selected for their very different degree of industrialization, urbanization, and conservation of local past traditions among the population: the Bologna province (Northern Italy) and the Middle Agri Valley (Southern Italy). Protein, polyphenol flavonoid and biogenic amine (both free and conjugated) contents and antioxidant activity of raw and boiled wild food plant extracts, and of cooking water were analyzed by means of spectrophotometric and high-performance liquid chromatography methods. The results demonstrated that most of the phenolic compounds were released in the cooking water which also showed the highest antioxidant activity. Seventeen different phenolic compounds were identified, of which the health-related luteolin, luteolin-7-glucoside and rutin were the most abundant (e.g., S. pratensis L. and C. intybus L.). On the other hand, biogenic amines were absent or present at very low levels in cooking water of those very same species (e.g., S. pratensis L., T. officinalis Weber, C. vesicaria subsp. taraxacifolia and C. intybus L.) of which traditionally a decoction is used for therapeutic purposes. Free and conjugated spermidine and spermine were generally the most abundant biogenic amines, while none of the known detrimental monoamines (e.g., histamine) was detected. In conclusion, the present results seem to support past local popular traditions which indicated beneficial medical properties of some wild edible plant, as well as of their cooking water.

Orthogonal paper biosensor for mercury(II) combining bioluminescence and colorimetric smartphone detection.

Mercury contamination in the environment has reached alarming levels. Due to its persistence and bioaccumulation, mercury is one of the most widespread toxic heavy metals found in air, water and food. Thus, it is mandatory to monitor mercury and its compounds, and the availability of sensitive and rapid biosensors is highly valuable. We developed a low-cost biosensor for orthogonal detection of mercury(II) integrating three different biorecognition principles on a three-leaf paper: i) a mercury-specific bioluminescent Escherichia coli bioreporter strain expressing NanoLuc luciferase as reporter protein, ii) a purified ß-galactosidase (ß-gal) enzyme which is irreversibly inhibited by mercury and other metal ions, and iii) an Aliivibrio fischeri bioluminescent strain which is used to quantitatively assess sample toxicity and correct the analytical signal accordingly. Both sensory elements and substrates, Furimazine for the bioluminescent reporter strain and chlorophenol red-ß-D-galactopyranoside for colorimetric detection of ß-gal, were integrated in the paper sensor to provide a stable all-in-one disposable cartridge which can be easily snapped into a smartphone with a clover-shaped 3D printed housing. This is the first integration of bioluminescence and colorimetric detection on a smartphone-paper sensor, providing a readout within 15 and 60 min for the colorimetric and bioluminescent detection respectively. The biosensor was applied to water samples spiked with different concentrations of mercury, interferents and toxic chemicals providing a limit of detection for Hg(II) at the ppb levels.

Enzymatic Digestion of Calf Fleshing Meat By-Products: Antioxidant and Anti-Tyrosinase Activity of Protein Hydrolysates, and Identification of Fatty Acids.

The food waste reduction through an efficient recovery of its valuable building molecules has become an important topic with a positive effect on the economy and the environment. In this work, the revalorization of slaughterhouse calf fleshing meat through its enzymatic hydrolysis is proposed. The proteolytic activity of 11 enzymes was initially screened and the four most efficient enzymes (papain, trypsin, pancreatin, and bromelain) were selected. The molecular profiling of the different protein/peptide fractions by the Linear Trap Quadrupole-OrbiTrap technique showed compositional differences due to the specificity of the enzymes' cleavage sites. In order to find a potential reuse of these hydrolysates, the analysis of antioxidant and, for the first time on fleshing meat hydrolysates, of anti-tyrosinase activities, was performed. Papain-digested samples were those showing the highest inhibition activity of tyrosinase enzyme (55.6%) as well as the highest antioxidant activity (3.52 g TEAC/L). In addition, the composition analysis of the lipid fraction was performed. The mono-unsaturated fatty acids resulted to be the most abundant lipid in all the samples with the exception of pancreatin-treated hydrolysates in which poly-unsaturated fatty acids were predominant. The present results seemed to support a possible valorization of isolated fractions from calf fleshing by-products, as food or feed ingredients, by the implementation of fraction isolation within the meat-processing pipeline.

Cascade strategies for the full valorisation of Garganega white grape pomace towards bioactive extracts and bio-based materials.

Agro-waste reduction and reuse are among the current main social challenges. In this perspective, the present research was aimed at the complete valorisation of Garganega grape pomace by recovering bioactive phenol extracts and by testing the solid fibre extract residues in composite formulation for packaging applications. The pomace was derived from white wine production, therefore, respect to red pomace, it was promptly removed from must after pressing, and its exploitation can be particularly interesting and valuable as still rich in active compounds. Phenol extracts were obtained both via solvent-based and pressurised liquid extractions and their phytochemical compositions were compared in terms of total amount of phenols, flavonoids, flavanols, anthocyanins, hydroxycinnamic acids, and reducing sugars. Antioxidant activity and detailed phenol profiles were also achieved. The highest phenol yield was obtained via solvent-based extraction with 75% acetone (v/v), solid/liquid ratio 1:5, 2h incubation at 50°C (77.9 gGAeq/kgDW). The fibrous solid residue of the extraction was characterized via thermogravimetric analysis and used for composite preparation by melt mixing with the renewable and biodegradable PHBV polymer through a green approach (solvent-less process). The composites resulted thermally stable at high temperatures, showing initial degradation processes only at temperatures higher than 250°C. Differential scanning calorimetry analyses were carried out to study melting and crystallization phenomena, while mechanical properties were investigated by tensile tests. The materials finally showed properties similar to those of the matrix. The bio-composites can be considered as an alternative to plain PHBV, since they are less expensive and eco-friendlier thanks to a reduced polymeric content, and they could represent a suitable way for full agro-waste exploitation.

Microbial Fermentation of Industrial Rice-Starch Byproduct as Valuable Source of Peptide Fractions with Health-Related Activity.

The rice-starch processing industry produces large amounts of a protein-rich byproducts during the conversion of broken rice to powder and crystal starch. Given the poor protein solubility, this material is currently discarded or used as animal feed. To fully exploit rice's nutritional properties and reduce this waste, a biotechnological approach was adopted, inducing fermentation with selected microorganisms capable of converting the substrate into peptide fractions with health-related bioactivity. Lactic acid bacteria were preferred to other microorganisms for their safety, efficient proteolytic system, and adaptability to different environments. Peptide fractions with different molecular weight ranges were recovered from the fermented substrate by means of cross-flow membrane filtration. The fractions displayed in vitro antioxidant, antihypertensive, and anti-tyrosinase activities as well as cell-based anti-inflammatory and anti-aging effects. In the future, the peptide fractions isolated from this rice byproduct could be directly exploited as health-promoting functional foods, dietary supplements, and pharmaceutical preparations. The suggested biotechnological process harnessing microbial bioconversion may represent a potential solution for many different protein-containing substrates currently treated as byproducts (or worse, waste) by the food industry.

From winery waste to bioactive compounds and new polymeric biocomposites: A contribution to the circular economy concept.

The paper aims at optimising and validating possible routes toward the full valorisation of grape agrowaste to produce bioactive molecules and new materials. Starting from Merlot red pomace, phenol complex mixtures were successfully extracted by using two different approaches. Extracts obtained by solvent-based (SE) technique contained up to 46.9 gGAeq/kgDW of total phenols. Depending on the used solvent, the prevalence of compounds belonging to different phenol families was achieved. Pressurized liquid extraction (PLE) gave higher total phenol yields (up to 79 gGAeq/kgDW) but a lower range of extracted compounds. All liquid extracts exerted strong antioxidant properties. Moreover, both SE and PLE extraction solid residues were directly exploited (between 5 and 20% w/w) to prepare biocomposite materials by direct mixing via an eco-friendly approach with PHBV polymer. The final composites showed mechanical characteristics similar to PHVB matrix. The use of pomace residues in biocomposites could therefore bring both to the reduction of the cost of the final material, as a lower amount of costly PHBV is used. The present research demonstrated the full valorisation of grape pomace, an agrowaste produced every year in large amounts and having a significant environmental impact.

Advances in combined enzymatic extraction of ferulic acid from wheat bran.

Wheat bran could be utilised as feedstock for innovative and sustainable biorefinery processes. Here, an enzymatic hydrolysis process for ferulic acid (FA) extraction was optimised step by step for total wheat bran (Tritello) and then also applied to the outer bran layer (Bran 1). Proteins, reducing sugars, total phenols and FA were quantified. The highest FA yields (0.82-1.05 g/kg bran) were obtained either by rehydrating the bran by autoclaving (Tritello) or by steam explosion (Bran 1) using a bran/water ratio of 1:20, followed by enzymatic pre-treatment with Alcalase and Termamyl, to remove protein and sugars, and a final enzymatic hydrolysis with Pentopan and feruloyl esterase to solubilise phenol. FA was recovered from the final digestate via solid phase extraction. A 40-fold scale-up was also performed and the release of compounds along all the process steps and at increasing incubation times was monitored. Results showed that FA was initially present at a minimum level while it was specifically released during the enzymatic treatment. In the final optimized process, the FA extraction yield was higher than that obtained with NaOH control hydrolysis while, in comparison with other FA enzymatic extraction methods, fewer process steps were required and no buffers, strong acid/alkali nor toxic compounds were used. Furthermore, the proposed process may be easily scaled-up, confirming the feasibility of wheat bran valorisation by biorefinery processes to obtain valuable compounds having several areas of potential industrial exploitation.

Enzymatic production of bioactive peptides from scotta, an exhausted by-product of ricotta cheese processing.

The present work reports the enzymatic valorisation of the protein fraction of scotta, a dairy by-product representing the exhausted liquid residue of ricotta production. Scotta was subjected to ultra-filtration with membrane cut-offs from 500 to 4 kDa and the obtained protein-enriched fractions were used for the optimization of enzyme-based digestions aimed at producing potentially bioactive peptides. Nine different commercial proteases were tested and the best digestion conditions were selected based on protein yield, fraction bioactivity and foreseen scale up processing costs. Scale up of the 3% Pancreatin or 5% Papain processes was performed up to 2 L (37°C or 60°C respectively, 1 h incubation), and the digestion efficiency increased with the reaction volume as well as antioxidant activity (up to 60 gBSA eq/L and to 1.7 gAA eq/L). Retentate 1 digested fractions also showed, for the first time in dairy-based peptides, anti-tyrosinase activity, up to 0.14 gKA eq/L. Digested proteins were sub-fractionated by means of physical membrane separations and 30-10 kDa fraction from Papain treatment showed the highest antioxidant and anti-tyrosinase activities. The peptide sequence of the most bioactive fractions was achieved.

Prêt-à-porter nanoYESα and nanoYESß bioluminescent cell biosensors for ultrarapid and sensitive screening of endocrine-disrupting chemicals.

Cell-based assays utilizing reporter gene technology have been widely exploited for biosensing, as they provide useful information about the bioavailability and cell toxicity of target analytes. The long assay time due to gene transcription and translation is one of the main drawbacks of cell biosensors. We report the development of two yeast biosensors stably expressing human estrogen receptors α and ß and employing NanoLuc as the reporter protein to upgrade the widely used yeast estrogen screening (YES) assays. A viability control strain was also developed based on a chimeric green-emitting luciferase, PLG2, expressed for the first time in Saccharomycescerevisiae. Thanks to their brightness, NanoLuc and PLG2 provided excellent sensitivity, enabling the implementation of these biosensors into low-cost smartphone-based devices. The developed biosensors had a rapid (1 h) response and reported on (anti)estrogenic activity via human estrogen receptors α and ß as well as general sample toxicity. Under optimized conditions, we obtained LODs of 7.1 ± 0.4 nM and 0.38 ± 0.08 nM for E2 with nanoYESα and nanoYESß, respectively. As a proof of concept, we analyzed real samples from plants showing significant estrogenic activity or known to contain significant amounts of phytoestrogens. Graphical abstract.

Publisher Correction: Bioprospecting on invasive plant species to prevent seed dispersal.

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Bioprospecting on invasive plant species to prevent seed dispersal.

ABSRACT: The most anthropized regions of the world are characterized by an impressive abundance of invasive plants, which alter local biodiversity and ecosystem services. An alternative strategy to manage these species could be based on the exploitation of their fruits in a framework of bioprospecting to obtain high-added value compounds or phytocomplexes that are useful for humans. Here we tested this hypothesis on three invasive plants (Lonicera japonica Thunb., Phytolacca americana L., and Prunus serotina Ehrh.) in the Po plain (northern Italy) which bear fruits that are highly consumed by frugivorous birds and therefore dispersed over large distances. Our biochemical analyses revealed that unripe fruit shows high antioxidant properties due to the presence of several classes of polyphenols, which have a high benchmark value on the market. Fruit collection for phytochemical extraction could really prevent seed dispersal mediated by frugivorous animals and produce economic gains to support local management actions.

Ethnobotanical survey of wild food plants traditionally collected and consumed in the Middle Agri Valley (Basilicata region, southern Italy).

BACKGROUND: This research was carried out in a scarcely populated area of the Middle Agri Valley (Basilicata region, southern Italy). The aim of the study was to record local knowledge on the traditional uses of wild food plants, as well as to collect information regarding the practices (gathering, processing and cooking) and the medicinal uses related to these plants. METHODS: Fifty-eight people still possessing traditional local knowledge (TLK), 74% women and 26% men, were interviewed between May-August 2012 and January 2013, using open and semi-structured ethnobotanical interviews. For each described plant species, the botanical family, the Italian common and folk names, the plant parts used, the culinary preparation and, when present, the medicinal use, were recorded and the relative frequency of citation index (RFC) was determined. RESULTS: The 52 plant species mentioned by the respondents belong to 23 botanical families, with Asteraceae (12 plants) and Rosaceae (7 plants) being most frequently cited. The species with the highest RFC index is Cichorium intybus L. (0.95), followed by Sonchus spp. (S. oleraceus L., S. asper L. and S. arvensis L.) (0.76). The plant parts preferably used are leaves (22 plants), fruits (12) and stems (7). Only six wild plants were indicated as having both food use and therapeutic effect. CONCLUSIONS: The survey conducted on the traditional use of wild food plants in the Middle Agri Valley revealed that this cultural heritage is only partially retained by the population. Over the last few decades, this knowledge has been in fact quickly disappearing along with the people and, even in the rural context of the study area, is less and less handed down to younger generations. Nevertheless, data also revealed that the use of wild plants is recently being revaluated in a way closely related to local habits and traditions.