Italian cider: a new potentially typical product to be characterized

The objective of this PhD research project has been to evaluate some characteristics (e.g., allergens content, polyphenols profile and antioxidant potential) of the Italian ciders that could affect the human health. In this way we tried to promote the product contributing to improve the knowledge needed to better the production techniques. The first aspect described in this PhD study was the recovery of cider proteins (Chapter 2). The rationale was the knowledge that, in other fermented beverages, these molecules are described to be involved in specific organoleptic characteristics such as foam stability, in the interaction with the aroma compounds in the formation of hazes and sediments. In addition, proteins could elicit allergenic reactions in sensitive individuals. After the setup of the recovery methods, proteins purified from 18 commercial ciders were identified by LC-MS/MS technique. This high sensitive method revealed the presence of Mal d 2 and Mal d 3 proteins in all the cider samples analyzed. In addition, this technique showed that 12 out of 18 ciders are contaminated by hen egg lysozyme that is a well-known allergen, even if its use and presence was not declared on the label as stated by the EU Directive. Since the LC-MS/MS analysis detects only fragments of protein that could not be immunologically active, ELISA and western blot analyses were achieved for the detection of lysozyme. Two samples out of 18 were both positive to the assays applied and this confirm the immunological activity of the protein and the existence of a serious health concern to drink cider for people sensitized to egg (Chapter 3). The following step was to study the effect of cidermaking procedure on apple allergen. For this reason, the immunological activity of Mal d 2 and Mal d 3 proteins was tested with specific antibodies developed in rabbit. Six and four samples out of the 13 ciders considered were positive to Mal d 2 and Mal d 3, respectively. It is important to highlight that not all the samples showed a positive match to both the apple allergens. This could indicate that the technological process in cidermaking could achieve a selective removal of specific allergens. Moreover, as regarding Mal d 3 the antibody detected an ambiguous signal at the level of the stacking gel of almost all samples (Chapter 4). Finally, an aspect related to the wholesomeness of cider was investigated. The polyphenols profiles of 18 Italian ciders were correlated with their antioxidant activity, and a multiple linear regression model was drawn to predict the antioxidant potential of the beverage, knowing the concentration of specific phenols (namely: chlorogenic acid, catechin, procyanidins B1 and B2, quercetins 3-glucoside, 3-xyloside and 3-arabinopyranoside). This suggests that these phenols could be the mainly contributors of the antioxidant activity, but further studies should be performed to exclude that this correlation could be only an epiphenomenon consequence of particular interactions among different molecules. Anyway the total antioxidant activities of Italian ciders results on average lower than those reported in literature for Asturian ones and this could be probably related to the total polyphenols content and their profiles (Chapter 5). This observation suggests that the Italian cidermaking process could be ameliorated taking in consideration both the allergenic profile of the beverage and the preferences of the Italian consumers

Phagocytosis of Astaxanthin-Loaded Microparticles Modulates TGFβ Production and Intracellular ROS Levels in J774A.1 Macrophages

Radiation-induced fibrosis is a serious long-lasting side effect of radiation therapy. Central to this condition is the role of macrophages that, activated by radiation-induced reactive oxygen species and tissue cell damage, produce pro-inflammatory cytokines, such as transforming growth factor beta (TGFb). This, in turn, recruits fibroblasts at the site of the lesion that initiates fibrosis. We investigated whether astaxanthin, an antioxidant molecule extracted from marine and freshwater organisms, could help control macrophage activation. To this purpose, we encapsulated food-grade astaxanthin from Haematococcus pluvialis into micrometer-sized whey protein particles to specifically target macrophages that can uptake material within this size range by phagocytosis. The data show that astaxanthin-loaded microparticles are resistant to radiation, are well-tolerated by J774A.1 macrophages, induce in these cells a significant reduction of intracellular reactive oxygen species and inhibit the release of active TGFb as evaluated in a bioassay with transformed MFB-F11 fibroblasts. Micro-encapsulation of bioactive molecules is a promising strategy to specifically target phagocytic cells and modulate their own functions

Effects of Combination Treatments with Astaxanthin-Loaded Microparticles and Pentoxifylline on Intracellular ROS and Radiosensitivity of J774A.1 Macrophages

Radiation-induced fibrosis (RIF) is a serious, yet incurable, complication of external beam radiation therapy for the treatment of cancer. Macrophages are key cellular actors in RIF because of their ability to produce reactive oxidants, such as reactive oxygen species (ROS) and inflammatory cytokines that, in turn, are the drivers of pro-fibrotic pathways. In a previous work, we showed that phagocytosis could be exploited to deliver the potent natural antioxidant astaxanthin specifically to macrophages. For this purpose, astaxanthin encapsulated into \u3bcm-sized protein particles could spe- cifically target macrophages that can uptake the particles by phagocytosis. In these cells, astaxanthin microparticles significantly reduced intracellular ROS levels and the secretion of bioactive TGF\u3b2 and increased cell survival after radiation treatments. Here we show that pentoxifylline, a drug currently used for the treatment of muscle pain resulting from peripheral artery disease, amplifies the effects of astaxanthin microparticles on J774A.1 macrophages. Combination treatments with pentoxifylline and encapsulated astaxanthin might reduce the risk of RIF in cancer patients

Influence of Tilia tomentosa Moench Extract on Mouse Small Intestine Neuromuscular Contractility

Functional gastrointestinal disorders (FGIDs) are characterized by abdominal pain, bloating and bowel disturbances. FGID therapy is primarily symptomatic, including treatment with herbal remedies. Flower extract of Tilia tomentosa Moench (TtM) is occasionally used as an anti-spasmodic in popular medicine. Since its effect on intestinal response is unknown, we evaluated the influence of TtM extract on small intestine contractility. Ileal preparations from C57BL/6J mice were mounted in organ baths to assess changes in muscle tension, following addition of TtM extract (0.5-36 mu g/mL) or a vehicle (ethanol). Changes in contractile response to receptor- and non-receptor-mediated stimuli were assessed in ileal preparations pretreated with 12 mu g/mL TtM. Alterations in the enteric nervous system neuroglial network were analyzed by confocal immunofluorescence. Increasing addition of TtM induced a marked relaxation in ileal specimens compared to the vehicle. Pretreatment with TtM affected cholinergic and tachykininergic neuromuscular contractions as well as K+-induced smooth muscle depolarization. Following incubation with TtM, a significant reduction in non-adrenergic non-cholinergic-mediated relaxation sensitive to N omega-Nitro-L-arginine methyl ester hydrochloride (pan-nitric oxide synthase inhibitor) was found. In vitro incubation of intestinal specimens with TtM did not affect the myenteric plexus neuroglial network. Our findings show that TtM-induced intestinal relaxation is mediated by nitric oxide pathways, providing a pharmacological basis for the use of TtM in FGIDs

Spray-drying Microencapsulation of an Extract from Tilia tomentosa Moench Flowers: Physicochemical Characterization and in Vitro Intestinal Activity

Silver linden (Tilia tomentosa Moench, TtM) flowers possess several health-promoting properties, especially at the neurological level, such as intestinal relaxation activity associated with specific flavonols, particularly quercetin and kaempferol derivatives. However, such molecules are susceptible to degradation upon different triggers like heat, light and extreme pH values. To overcome the scarce stability of TtM flowers bioactive molecules and make them suitable for developing functional food and supplements, we applied microencapsulation. Spray-drying microencapsulation of TtM flowers extract was performed using three starch-derived wall materials: maltodextrin 12 DE (MD12) and 19 DE (MD19), and OSA-modified starch (OSA-S). The stability of total phenols, flavanols, and antioxidant capacity was monitored for 70 days under accelerated stress conditions (40 °C/70% RH) by HPLC and spectrophotometric methods, and the intestinal contractile activity was tested in a murine model. In comparison to MD12 and MD19, OSA-S stood out for the higher encapsulation efficiency of quercetin and kaempferol glycosides (+ 36-47% compared to MD12 and + 18-24% compared to MD19) and stability thereof (half-life on average + 30% compared to MD12 and + 51% compared to MD19). The intestinal contractile activity of OAS-S powders resulted comparable to the original extract, indicating that flavonols were biologically active and accessible. Our results underly the potential advantages of OSA-S encapsulated formulation as a functional ingredient for the development of nutraceutical products

Italian cider: a new potentially typical product to be characterized

The objective of this PhD research project has been to evaluate some characteristics (e.g., allergens content, polyphenols profile and antioxidant potential) of the Italian ciders that could affect the human health. In this way we tried to promote the product contributing to improve the knowledge needed to better the production techniques. The first aspect described in this PhD study was the recovery of cider proteins (Chapter 2). The rationale was the knowledge that, in other fermented beverages, these molecules are described to be involved in specific organoleptic characteristics such as foam stability, in the interaction with the aroma compounds in the formation of hazes and sediments. In addition, proteins could elicit allergenic reactions in sensitive individuals. After the setup of the recovery methods, proteins purified from 18 commercial ciders were identified by LC-MS/MS technique. This high sensitive method revealed the presence of Mal d 2 and Mal d 3 proteins in all the cider samples analyzed. In addition, this technique showed that 12 out of 18 ciders are contaminated by hen egg lysozyme that is a well-known allergen, even if its use and presence was not declared on the label as stated by the EU Directive. Since the LC-MS/MS analysis detects only fragments of protein that could not be immunologically active, ELISA and western blot analyses were achieved for the detection of lysozyme. Two samples out of 18 were both positive to the assays applied and this confirm the immunological activity of the protein and the existence of a serious health concern to drink cider for people sensitized to egg (Chapter 3). The following step was to study the effect of cidermaking procedure on apple allergen. For this reason, the immunological activity of Mal d 2 and Mal d 3 proteins was tested with specific antibodies developed in rabbit. Six and four samples out of the 13 ciders considered were positive to Mal d 2 and Mal d 3, respectively. It is important to highlight that not all the samples showed a positive match to both the apple allergens. This could indicate that the technological process in cidermaking could achieve a selective removal of specific allergens. Moreover, as regarding Mal d 3 the antibody detected an ambiguous signal at the level of the stacking gel of almost all samples (Chapter 4). Finally, an aspect related to the wholesomeness of cider was investigated. The polyphenols profiles of 18 Italian ciders were correlated with their antioxidant activity, and a multiple linear regression model was drawn to predict the antioxidant potential of the beverage, knowing the concentration of specific phenols (namely: chlorogenic acid, catechin, procyanidins B1 and B2, quercetins 3-glucoside, 3-xyloside and 3-arabinopyranoside). This suggests that these phenols could be the mainly contributors of the antioxidant activity, but further studies should be performed to exclude that this correlation could be only an epiphenomenon consequence of particular interactions among different molecules. Anyway the total antioxidant activities of Italian ciders results on average lower than those reported in literature for Asturian ones and this could be probably related to the total polyphenols content and their profiles (Chapter 5). This observation suggests that the Italian cidermaking process could be ameliorated taking in consideration both the allergenic profile of the beverage and the preferences of the Italian consumers.L'obiettivo di questa tesi di dottorato è stato quello di valutare alcune caratteristiche dei sidri Italiani che potrebbero incidere sulla salute umana, come ad esempio, gli allergeni, il contenuto di polifenoli e il potenziale antiossidante, incrementando in questo modo il valore aggiunto della bevanda. In un certo senso, si è cercato di promuovere la commercializzazione del prodotto, contribuendo a migliorare le conoscenze necessarie per ottimizzarne le tecniche di produzione. Il primo aspetto descritto in questo studio è stato la purificazione preparativa delle proteine del sidro (Capitolo 2). La logica di questo approccio è costituita dalla conoscenza che, in altre bevande fermentate, queste molecole sono descritte come coinvolte nel determinare caratteristiche organolettiche specifiche, quali la stabilità della schiuma, l'interazione con i composti aromatici e la formazione di intorbidamenti e di sedimenti. Va inoltre tenuto presente che, le proteine del sidro potrebbero essere coinvolte nello scatenamento di reazioni allergiche in soggetti sensibili. Dopo la messa a punto dei metodi preparativi, le proteine purificate da 18 sidri commerciali sono state identificate con la tecnica LC-MS/MS. Questo metodo ad alta sensibilità ha rivelato la presenza delle proteine Mal d 2 e Mal d 3 in tutti i campioni analizzati. Inoltre, questa tecnica ha dimostrato come 12 sidri su 18 campioni siano contaminati da lisozima di uovo di gallina. Quest’ultima proteina è un noto allergene, anche se la sua presenza non risultava dichiarata in etichetta, come invece sarebbe previsto dalla Direttiva dell'Unione Europea 2007/68/CE. Poiché l'analisi LC-MS/MS rileva solo frammenti di proteina che potrebbero non essere immunologicamente attivi, sono stati eseguiti un test ELISA e l’analisi con la metodica western blot. Due campioni su 18 sono risultati positivi ai test applicati e questo ha confermato l'attività immunologica della proteina e l'esistenza di un serio rischio per la salute delle persone sensibilizzate alle uova, qualora dovessero bere del sidro (Capitolo 3). Il passo successivo è stato quello di studiare l'effetto della procedura di produzione del sidro sugli allergeni della mela. Per questo motivo, l'attività immunologica delle proteine Mal d 2 e Mal d 3 è stata testata con anticorpi (IgG) specifici sviluppati in coniglio. Rispettivamente sei e quattro sidri su 13 campioni sono risultati positivi per Mal d 2 e Mal d 3. È importante sottolineare che non tutti i campioni hanno mostrato un risultato positivo per entrambi questi allergeni di mela. Questo indicherebbe che alcuni passaggi del processo tecnologico di produzione del sidro potrebbero produrre una rimozione selettiva di specifici allergeni. Tuttavia, riguardo alla proteina Mal d 3, il saggio anticorpale in western blot, ha rilevato un segnale ambiguo a livello del gel di impaccamento di quasi tutti i campioni (Capitolo 4). Questo dato dovrà essere oggetto di ulteriori approfondimenti. Infine, è stato studiato un aspetto che è correlato alla salubrità del sidro. I profili polifenolici di 18 sidri Italiani sono stati correlati con la loro attività antiossidante, ed è stato realizzato un modello di regressione lineare multipla al fine di correlare il potenziale antiossidante della bevanda con la concentrazione di polifenoli specifici (in particolare: acido clorogenico, catechina, procianidine B1 e B2, quercetine 3-glucoside, 3-xiloside e 3-arabinopiranoside). I risultati ottenuti potrebbero suggerire che questi polifenoli siano quelli che principalmente contribuiscono all’attività antiossidante della bevanda, ma ulteriori studi dovranno essere eseguiti per escludere che questa correlazione non possa essere solo un epifenomeno dovuto, per esempio, a particolari interazioni di molecole diverse. In ogni caso, l’attività antiossidante dei sidri Italiani è risultata mediamente inferiore a quella riportata in letteratura per i sidri delle Asturie, e questo potrebbe probabilmente essere dovuto al contenuto di polifenoli totali e al loro profilo (Capitolo 5). Tutte queste osservazioni suggeriscono che il sidro, attualmente prodotto in Italia, sia un prodotto che potrebbe essere migliorato sotto il punto di vista salutistico pur tenendo in considerazione le peculiarità organolettiche ricercate dal consumatore locale

Rheology of individual chitosan and polyphenol/chitosan microparticles for food engineering

Encapsulation of perishable bioactive molecules has become a standard process in the food industry. Beads in the millimeter size range may offer technological advantage over smaller particles and nano-structured materials for encapsulation purposes because of higher volume to surface ratios, but their size might seriously affect the sensory properties of the final edible products. We investigated the rheological properties of individual beads composed of chitosan (CTS) cross-linked with sodium tripolyphosphate (TTP). We produced the beads by the external gelation method using different combinations of CTS and TTP concentrations. Analysis of raw data showed that CTS and TTP affected both the morphology and the response of the particles to mechanical load in a non proportional way. The data indicated that the mechanical properties of the beads could not be explained with the aid of standard linear viscoelastic models. We therefore exploited a recently-developed nonlinear model to analyse mechanical data collected during the stress phase and estimate viscoelastic parameters. We found a clear synergic effect of CTS and TTP concentrations on particles’ stiffness and a crossover effect on their viscosity. Two representative bioactive food phenolic compounds, i.e. chlorogenic acid and catechin, were then encap- sulated into the beads. Encapsulation of chlorogenic acid and catechin dramatically altered the viscoelastic behaviour of the particles in a similar way. The particles became less stiff and viscous and more fluid-like compared to empty beads with the same CTS and TTP composition. Our results pave the way for a more ac- curate evaluation of sensory characteristics of novel functional foods incorporating particle carriers for bioactive molecules

Allergy to pomegranate

Food allergy is an immunological disease whose prevalence is increasing worldwide. Pomegranate could determine allergic reactions, even if a limited number of cases is reported in literature. In the present chapter, after an introductive part describing the pathogenesis and the clinical aspects of allergy and more specifically food allergy, we present a review of the scientific literature dealing with allergy to pomegranate, examining more in depth the involvement of non specific Lipid Transfer Proteins (nsLTP). Indeed, these proteins represent the only pomegranate allergens characterized at the molecular level