Sviluppo di un metodo elettrochimico per la determinazione della capacità antiossidante

The aim of the present thesis, carried out at the Analytical Group of the Faculty of Industrial Chemistry in Bologna, is to develop a new electrochemical method for the determination of the Antioxidant Capacity (AOC). The approach is based on the deposition of a non-conducting polymeric film on the working electrode surface and the following exposition to the radicals OH· produced by H2O2 photolysis. The strongly oxidant action of hydroxyl radicals degrades, causing an increase of the Faradic current, relevant to the redox couple [Ru(NH3)6]2+/3+ monitored by cyclic voltammetry(CV); the presence of an antioxidant compound in solution slows down the radical action, thus protecting the polymeric film and blocking the charge transfer. The parameter adopted for the quantification of the AOC, was the induction time, called also lag phase, which is the time when the degradation of the film starts. Five pure compounds, among most commonly antioxidant, were investigated : Trolox®(an analogue water-soluble of vitamin E), (L)-ascorbic acid, gallic acid, pyrogallol and (-)- epicatechin. The AOC of each antioxidant was expressed by TEAC index (Trolox® Equivalent Antioxidant Capacity), calculated from the ratio between the slope of the calibration curve of the target compound and the slope of the calibration curve of Trolox®. The results from the electrochemical method, have been compared with those obtained from some other standardized methods, widely employed. The assays used for the comparison, have been: ORAC, a spectrofluorimetric method based on the decrease of fluorescein emission after the attack of alkylperoxide radicals, ABTS and DPPH that exploit the decoloration of stable nitrogen radicals when they are reduced in presence of an antioxidant compound and, finally, a potentiometric method based on the response of the redox couple [Fe(CN)6]3-/ [Fe(CN)6]4-. From the results obtained from pure compounds, it has been found that ORAC is the methodology showing the best correlation with the developed electrochemical method, maybe since similar radical species are involved. The comparison between the considered assays, was also extended to the analysis of a real sample of fruit juice. In such a case the TEAC value resulting from the electrochemical method is higher than those from standardized assays.

Development of instrumental and sensory analytical methods of food obtained by traditional and emerging technologies

The consumer demand for natural, minimally processed, fresh like and functional food has lead to an increasing interest in emerging technologies. The aim of this PhD project was to study three innovative food processing technologies currently used in the food sector. Ultrasound-assisted freezing, vacuum impregnation and pulsed electric field have been investigated through laboratory scale systems and semi-industrial pilot plants. Furthermore, analytical and sensory techniques have been developed to evaluate the quality of food and vegetable matrix obtained by traditional and emerging processes. Ultrasound was found to be a valuable technique to improve the freezing process of potatoes, anticipating the beginning of the nucleation process, mainly when applied during the supercooling phase. A study of the effects of pulsed electric fields on phenol and enzymatic profile of melon juice has been realized and the statistical treatment of data was carried out through a response surface method. Next, flavour enrichment of apple sticks has been realized applying different techniques, as atmospheric, vacuum, ultrasound technologies and their combinations. The second section of the thesis deals with the development of analytical methods for the discrimination and quantification of phenol compounds in vegetable matrix, as chestnut bark extracts and olive mill waste water. The management of waste disposal in mill sector has been approached with the aim of reducing the amount of waste, and at the same time recovering valuable by-products, to be used in different industrial sectors. Finally, the sensory analysis of boiled potatoes has been carried out through the development of a quantitative descriptive procedure for the study of Italian and Mexican potato varieties. An update on flavour development in fresh and cooked potatoes has been realized and a sensory glossary, including general and specific definitions related to organic products, used in the European project Ecropolis, has been drafted.

Isolierung und Identifizierung von wachstumsfördernden Substanzen für das weinrelevante Bakterium Oenococcus oeni aus Fruchtsäften und Blattextrakten

Neben Tomatensaft wurde eine Vielzahl von Säften und Blattextrakten als Medienzusätze auf Wachstumsförderung bei 30 verschiedenen Oenococcus oeni-Stämmen getestet. Es zeigte sich eine breite Wachstumsförderung bei allen Zusätzen mit Ausnahme von Zitronensaft, sodass die Wachstumsfaktoren keine tomatenspezifischen Inhaltsstoffe sein können und eher ubiquitär in der Pflanzenwelt vorkommen. Das Ausmaß der Wachstumsförderung war stammabhängig sehr unterschiedlich und Tomatensaft stellte keineswegs für alle Stämme den optimalen Medienzusatz dar. Durch Schälen der Früchte war eine für die Analytik hilfreiche Abtrennung schalenspezifischer Inhaltsstoffe möglich, wobei auch die Schalenextrakte großes Potential für die Suche nach Wachstumsfaktoren offenbarten und die Wichtigkeit einer Auftrennung der Frucht in die verschiedenen Fruchtbereiche betonte. Aus Tomatensaft konnte analytisch der anorganische Wachstumsfaktor Mangan identifiziert werden. Die größten Zelldichten der Oenokokken-Stämme wurden hierbei bei 67 µM und 34 mM Manganzusatz erreicht. Bei 13 von 20 getesteten Oenokokkenstämmen konnte bei Zusatz von 34 mM Mangan der Tomatensaft ersetzt werden, bei 4 Stämmen (z. B. Stamm B2) fehlten jedoch noch weitere Wachstumsfaktoren und bei 3 Stämmen (z. B. Stamm B120) kam es zu einem verfrühten Absterben. Da weitere Mineralstoffe sowie veraschte Säfte und Blattextrakte keinen positiven Einfluß auf die Oenokokken-Zelldichte hatten, wurde mittels semipräparativer HPLC nach zusätzlichen organischen Wachstumsfaktoren für den Stamm B2 gesucht. Hierzu wurde der nachfolgende Wachstums-Assay miniaturisiert und erfolgreich auf Microtiterplatten etabliert. Es gelang die Isolierung und Identifizierung eines wachstumsfördernden Trisaccharides aus Mangoschalen-Extrakt, das aus den Zuckern Glucose, Rhamnose und Arabinose bestand. Von den monomeren Zuckern erhöhte lediglich die Arabinose die Zelldichte, das Optimum lag bei 1,5 g/l. Auch aus Zitronenmesokarp-Extrakt war die Isolierung eines wachstumsfördernden arabinosehaltigen Disaccharides möglich, die Menge reichte jedoch noch nicht für eine genaue Identifizierung aus. Desweiteren erwies sich 1,5 g/l Cystein als wachstumsstimulierend. Ein Zusatz aller gefundenen Wachstumsfaktoren (34 mM Mangan, 1,5 g/l Arabinose und 1,5 g/l Cystein) ersetzte den Tomatensaft bei weiteren Oenokokken-Stämmen (z.B. Stamm B120) komplett, wobei bei allen Stämmen sogar eine schnellere Anzucht erfolgte. Neben dem Tomatensaft war auch der Zusatz von Hefeextrakt zum Grundmedium nicht mehr nötig, sodass ein neues vereinfachtes Medium für die Anzucht von Oenokokken mit komplexen Nährstoffansprüchen vorgeschlagen werden konnte. Lediglich beim Stamm B2 zeigte sich noch ein OD-Unterschied von 0,2 in der stationären Phase, der nach Adsorptionsversuchen an Polyvinylpolypyrrolidon auf noch unidentifizierte Polyphenole im Tomatensaft zurückzuführen ist. Aus grünem Tee erwies sich das Polyphenol Epigallocatechingallat (EGCG) konzentrationsabhängig sowohl als Hemmstoff (>550 mg/l EGCG) als auch Wachstumsfaktor (400-500 mg/l EGCG) für den Oenokokken-Stamm B2. Der hemmende als auch der fördernde Einfluss auf das Wachstum wurde mittels Sytox/DAPI-Färbung bestätigt. Der sogenannte „Tomatensaft-Faktor“ ist also nicht eine spezielle Substanz, sondern das synergistische Zusammenwirken mehrerer einfacher Substanzen wie Mineralstoffe, Aminosäuren, Kohlenhydrate und Polyphenole. Auch sind die Oenokokken-Stämme bezüglich ihres Nährstoffbedarfes sehr unterschiedlich, sodass für jeden Stamm einzeln das optimale Substratspektrum ermittelt werden muss.

1st and 2nd Generation Ethanol from Biomass Crops

In chapter 1 and 2 calcium hydroxide as impregnation agent before steam explosion of sugarcane bagasse and switchgrass, respectively, was compared with auto-hydrolysis, assessing the effects on enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) at high solid concentration of pretreated solid fraction. In addition, anaerobic digestion of pretreated liquid fraction was carried out, in order to appraise the effectiveness of calcium hydroxide before steam explosion in a more comprehensive way. In As water is an expensive input in both cultivation of biomass crops and subsequent pretreatment, Chapter 3 addressed the effects of variable soil moisture on biomass growth and composition of biomass sorghum. Moreover, the effect of water stress was related to the characteristics of stem juice for 1st generation ethanol and structural carbohydrates for 2nd generation ethanol. In the frame of chapter 1, calcium hydroxide was proven to be a suitable catalyst for sugarcane bagasse before steam explosion, in order to enhance fibre deconstruction. In chapter 2, effect of calcium hydroxide on switchgrass showed a great potential when ethanol was focused, whereas acid addition produced higher methane yield. Regarding chapter 3, during crop cycle the amount of cellulose, hemicellulose and AIL changed causing a decrease of 2G ethanol amount. Biomass physical and chemical properties involved a lower glucose yield and concentration at the end of enzymatic hydrolysis and, consequently, a lower 2G ethanol concentration at the end of simultaneous saccharification and fermentation, proving that there is strong relationship between structure, chemical composition, and fermentable sugar yield. The significantly higher concentration of ethanol at the early crop stage could be an important incentive to consider biomass sorghum as second crop in the season, to be introduced into some agricultural systems, potentially benefiting farmers and, above all, avoiding the exacerbation of the debate about fuel vs food crops.

Mikrobielle Synthese von Biopolymeren aus der nachwachsenden Rohstoffquelle Weizenstroh

Weizenstroh als erneuerbare Ressource zur Produktion von Biopolymeren und wichtigen Grundchemikalien stellt eine ökologisch sinnvolle Alternative dar. Durch die vom PFI durchgeführte Thermodruckhydrolyse konnte das Weizenstroh und die darin enthaltenen Zucker fast vollständig mobilisiert werden. Ein umfangreiches Screening nach Organismen, welche die Zucker des Weizenstrohs verwerten konnten, ergab, dass einige wenige Stämme zur PHB-Bildung aus Xylose befähigt waren (10 %). Zur PHB-Synthese aus Glucose waren indes ca. doppelt so viele Organismen in der Lage (20 %). Zwei der insgesamt 118 untersuchten Organismen zeigten besonders gute PHB-Bildung sowohl mit Xylose als auch mit Glucose als Substrat. Dabei handelte es sich um die hauseigenen Stämme Bacillus licheniformis KHC 3 und Bacillus megaterium KNaC 2. Nach Enttoxifizierung der hemicellulosischen Fraktion konnte diese als C-Quelle im Mineral Medium eingesetzt werden. Burkholderia sacchari DSM 17165 und Hydrogenophaga pseudoflava DSM 1034, sowie die hauseigenen Isolate Bacillus licheniformis KHC 3 und Bacillus megaterium KNaC 2 wurden für die Synthese von PHB aus der hemicellulosischen Fraktion verwendet. Die Zucker der hemicellulosischen Fraktion (Xylose, Glucose, Arabinose) konnten durch diese Organismen zur PHB-Synthese genutzt werden. Hierbei stellte sich heraus, dass die beiden Bacillus-Stämme besser zur Produktion von PHB aus dem hemicellulosischen Hydrolysat geeignet waren als die Stämme der DSMZ. Die alternative Umsetzung der im hemicellulosischem Hydrolysat enthaltenen Zucker (Xylose, Glucose und Arabinose) in die wichtigen Grundchemikalien Lactat und Acetat konnte durch die Verwendung von heterofermentativen Milchsäurebakterien verwirklicht werden. Die Bildung dieser wichtigen Grundchemikalien stellt eine interessante Alternative zur PHB-Synthese dar. Die Menge an teuren Zusätzen wie Tomatensaft, welcher für das Wachstum der MSB essentiell war, konnte reduziert werden. Die Glucose der zweiten Fraktion des Weizenstrohs, der cellulosischen Fraktion, konnte ebenfalls durch den Einsatz von Mikroorganismen in PHB umgewandelt werden. Kommerzielle Cellulasen der Firma Novozymes konnten große Mengen an Glucose (≥10 g/l) aus der cellulosischen Fraktion freisetzen. Diese freie Glucose wurde mit Hilfe von Cupriavidus necator DSM 545, Cupriavidus necator NCIMB 11599, Bacillus licheniformis KHC 3 und Bacillus megaterium KNaC 2 zu PHB fermentiert. Wie auch beim hemicellulosischen Hydrolysat konnten hier die beiden Bacillus-Stämme die besten Ergebnisse erzielen. Bei ihnen machte die PHB mehr als die Hälfte der Trockenmasse aus. Die Abtrennung des Zielprodukts ohne die Verwendung von umweltschädlichen Lösungsmitteln wurde durch die Lyse der Zielzellen durch eigens isolierte Enzyme aus Streptomyceten verwirklicht. Die Zelllyse durch die Enzyme aus Streptomyces globisporus subsp. caucasius DSM 40814 und Streptomyces albidoflavus DSM 40233 war erfolgreich und zeigte vor allem bei den Bacillen hohe Wirkung (83 % und 99 % Zelllyse). Bei dem Gram-negativen Organismus Cupriavidus necator DSM 428 konnte die anfangs niedrige Zelllyse von 38 % durch Ultraschallbehandlung auf ca. 75 % erhöht werden.

Thickness of softened human enamel removed by toothbrush abrasion: an in vitro study

The aim of the study was to assess the thickness of softened enamel removed by toothbrushing. Human enamel specimens were indented with a Knoop diamond. Softening was performed with citric acid or orange juice. The specimens were brushed in a brushing machine with a manual soft toothbrush in toothpaste slurry or in artificial saliva. Enamel loss was calculated from the change in indentation depth of the same indent before and after abrasion. Mean surface losses (95% confidence interval) were recorded in treatment groups (in nanometers): (1) citric acid, abrasion with slurry = 339 (280-398); (2) citric acid, abrasion with artificial saliva = 16 (5-27); (3) orange juice, abrasion with slurry = 268 (233-303); (4) orange juice, abrasion with artificial saliva = 14 (5-23); (5) no softening, abrasion with slurry = 28 (10-46). The calculated thickness of the softened enamel varied between 254 and 323 nm, depending on the acid used.

Analysis of the erosive effect of different dietary substances and medications

Excessive consumption of acidic drinks and foods contributes to tooth erosion. The aims of the present in vitro study were twofold: (1) to assess the erosive potential of different dietary substances and medications; (2) to determine the chemical properties with an impact on the erosive potential. We selected sixty agents: soft drinks, an energy drink, sports drinks, alcoholic drinks, juice, fruit, mineral water, yogurt, tea, coffee, salad dressing and medications. The erosive potential of the tested agents was quantified as the changes in surface hardness (ΔSH) of enamel specimens within the first 2 min (ΔSH2-0 = SH2 min - SHbaseline) and the second 2 min exposure (ΔSH4-2 = SH4 min - SH2 min). To characterise these agents, various chemical properties, e.g. pH, concentrations of Ca, Pi and F, titratable acidity to pH 7·0 and buffering capacity at the original pH value (β), as well as degree of saturation (pK - pI) with respect to hydroxyapatite (HAP) and fluorapatite (FAP), were determined. Erosive challenge caused a statistically significant reduction in SH for all agents except for coffee, some medications and alcoholic drinks, and non-flavoured mineral waters, teas and yogurts (P < 0·01). By multiple linear regression analysis, 52 % of the variation in ΔSH after 2 min and 61 % after 4 min immersion were explained by pH, β and concentrations of F and Ca (P < 0·05). pH was the variable with the highest impact in multiple regression and bivariate correlation analyses. Furthermore, a high bivariate correlation was also obtained between (pK - pI)HAP, (pK - pI)FAP and ΔSH.

Preventing erosion with novel agents

Objectives: This in vitro study aimed to investigate the protective effect of four commercial novel agents against erosion. Methods: Ninety human molars were distributed into 9 groups, and after incubation in human saliva for 2 h, a pellicle was formed. Subsequently, the specimens were submitted to demineralization (orange juice, pH 3.6, 3 min) and remineralization (paste slurry containing one of the tested novel agents, 3 min) cycles, two times per day, for 4 days. The tested agents were: (1) DenShield Tooth; active ingredient: 7.5% W/W NovaMin® (calcium sodium phosphosilicate); (2) Nanosensitive hca; active ingredient: 7.5% W/W NovaMin®; (3) GC Tooth Mousse; active ingredient: 10% Recaldent™ (CPP-ACP); (4) GC MI Paste Plus; active ingredients: 10% Recaldent™, 900 ppm fluoride. Two experimental procedures were performed: in procedure 1, the tested agents were applied prior to the erosive attack, and in procedure 2 after the erosive attack. A control group receiving no prophylactic treatment was included. Surface nanohardness (SNH) of enamel specimens was measured after pellicle formation and after completion of daily cyclic treatment. Results: SNH significantly decreased at the end of the experiment for all groups (p < 0.05). In both procedures, there was no statistically significant difference between the control group and those treated with paste slurries (p > 0.05). In addition, the changes in SNH (ΔSNH = SNHbaseline − SNHfinal) did not show statistically significant difference between both procedures (p > 0.05). Conclusion: Tooth erosion cannot be prevented or repaired by these novel agents, regardless of fluoride content.

Investigation of different apple cultivars by high resolution magic angle spinning NMR. A feasibility study

(1)H HR-MAS NMR spectroscopy was applied to apple tissue samples deriving from 3 different cultivars. The NMR data were statistically evaluated by analysis of variance (ANOVA), principal component analysis (PCA), and partial least-squares-discriminant analysis (PLS-DA). The intra-apple variability of the compounds was found to be significantly lower than the inter-apple variability within one cultivar. A clear separation of the three different apple cultivars could be obtained by multivariate analysis. Direct comparison of the NMR spectra obtained from apple tissue (with HR-MAS) and juice (with liquid-state HR NMR) showed distinct differences in some metabolites, which are probably due to changes induced by juice preparation. This preliminary study demonstrates the feasibility of (1)H HR-MAS NMR in combination with multivariate analysis as a tool for future chemometric studies applied to intact fruit tissues, e.g. for investigating compositional changes due to physiological disorders, specific growth or storage conditions.

Clinical Study Monitoring the pH on Tooth Surfaces in Patients with and without Erosion

The aim of this study was to compare tooth surface pH after drinking orange juice or water in 39 patients with dental erosion and in 17 controls. The following investigations were carried out: measurement of pH values on selected tooth surfaces after ingestion of orange juice followed by ingestion of water (acid clearance), measurement of salivary flow rate and buffering capacity. Compared with the controls, patients with erosion showed significantly greater decreases in pH after drinking orange juice, and the pH stayed lower for a longer period of time (p < 0.05). Saliva parameters showed no significant differences between the two patient groups except for a lower buffering capacity at pH 5.5 in the erosion group.

Exploring High-resolution Magic Angle Spinning (HR-MAS) NMR Spectroscopy for Metabonomic Analysis of Apples

Classical liquid-state high-resolution (HR) NMR spectroscopy has proved a powerful tool in the metabonomic analysis of liquid food samples like fruit juices. In this paper the application of (1)H high-resolution magic angle spinning (HR-MAS) NMR spectroscopy to apple tissue is presented probing its potential for metabonomic studies. The (1)H HR-MAS NMR spectra are discussed in terms of the chemical composition of apple tissue and compared to liquid-state NMR spectra of apple juice. Differences indicate that specific metabolic changes are induced by juice preparation. The feasibility of HR-MAS NMR-based multivariate analysis is demonstrated by a study distinguishing three different apple cultivars by principal component analysis (PCA). Preliminary results are shown from subsequent studies comparing three different cultivation methods by means of PCA and partial least squares discriminant analysis (PLS-DA) of the HR-MAS NMR data. The compounds responsible for discriminating organically grown apples are discussed. Finally, an outlook of our ongoing work is given including a longitudinal study on apples.

Bryophyllum pinnatum inhibits detrusor contractility in porcine bladder strips--a pharmacological study towards a new treatment option of overactive bladder

A broad spectrum of synthetic agents is available for the treatment of overactive bladder. Anti-cholinergic drugs show a poor compliance due to side effects. There is an increasing use of plant extracts in medicine. We have therefore investigated the inhibitory effects of leaf press juice from Bryophyllum pinnatum (Lam.) Oken (Kalanchoe pinnata L.) on bladder strips and compared the effects to that of oxybutynin.

Effects of expectation and caffeine on arousal, well-being, and reaction time

The objective of this study is to determine the impact of expectation associated with placebo and caffeine ingestion. We used a three-armed, randomized, double-blind design. Two three-armed experiments varying instruction (true, false, control) investigated the role of expectations of changes in arousal (blood pressure, heart rate), subjective well-being, and reaction time (RT). In Experiment 1 (N = 45), decaffeinated coffee was administered, and expectations were produced in one group by making them believe they had ingested caffeinated coffee. In Experiment 2 (N = 45), caffeinated orange juice was given in both experimental groups, but only one was informed about the true content. In Experiment 1, a significant effect for subjective alertness was found in the placebo treatment compared to the control group. However, for RT and well-being no significant effects were found. In Experiment 2, no significant expectancy effects were found. Caffeine produced large effects for blood pressure in both treatments compared to the control group, but the effects were larger for the false information group. For subjective well-being (alertness, calmness), considerable but nonsignificant changes were found for correctly informed participants, indicating possible additivity of pharmacologic effect and expectations. The results tentatively indicate that placebo and expectancy effects primarily show through introspection.

Isolated extraordinary daytime urinary frequency of childhood: a case series of 26 children in Switzerland

BACKGROUND: The term 'isolated extraordinary daytime urinary frequency' designates an abnormally increased diurnal frequency of painless urination in a completely toilet-trained child with normal urinalysis. METHODS: We report the history of 26 children (16 boys and 10 girls; aged between 4.1 and 10 years) who were referred to us between 2002 and 2006 and subsequently diagnosed with this condition. RESULTS: Possible psychosocial problems, or recent emotional stressors, were disclosed in the majority of the children: recent (36 months or less) asylum seekers (n = 9), school-related problems (n = 4), parental divorce (n = 2) or death of the mother (n = 1). Possible dietary causes were observed in 9 patients: oxalate-rich beverages (n = 5) and liberal ingestion of 'acidic' juices (n = 4). A diet low in oxalates was recommended when children were consuming large quantities of oxalate-rich beverages; and a diet low in acidic juice was recommended in those liberally ingesting acidic juices. Reassurance and observation were the approach in the remaining cases. The median duration of the symptoms was 5 months. A longer (p < 0.05) duration was noted in children of asylum seekers. CONCLUSIONS: This functional condition is easily identifiable, but often under- or misdiagnosed. Confounding the condition might result in redundant investigation.

[Occurrence of Trichinella spp. in wild boar in Switzerland]

Trichinellosis is a worldwide occurring zoonosis caused by the intracellular nematode Trichinella spp. One of the main infection sources in Europe is raw or undercooked meat from wild boar. Trichinella britovi is prevalent in wild carnivores in Switzerland, thus a possible inclusion of wild boar in this wildlife cycle cannot be excluded. In order to assess the prevalence of Trichinella infection in wild boar, we tested 1,458 animals with both parasitological and serological methods. In none of the animals Trichinella-larvae could be recovered by the artificial digestion method (prevalence of larvae: 0 %; 95 % CI 0.0 - 0.3). Antibodies in meat juice were detected in 57 animals using a standardized E/S-Ag-ELISA. However, in the confirmatory westernblot, only 3 animals remained seropositive (seroprevalence: 0.2 %; 95 % CI 0.07 %-0.60 %). The occurrence of wild boar positive for anti-Trichinella-antibodies indicates that meat inspection for Trichinella-larvae in this species is important to prevent human infections.