Interregional and interannual variability of ethanol site-specific deuterium content in Italian wines

The site-specific natural isotope fractionationnuclear magnetic resonance (SNIF-NMR)²H analysis is at present the reference method for determining the origin of ethanol and has been successfully applied for analysing all grape products. The method has also been used to identify the origin of wines and the aim of this study was to verify this application. In all, 3309 samples belonging to the Italian wines data bank were analysed from all 20 Italian regions to study the possibilities of determining and controlling the exact origin and year of the product. The dependency of the deuterium/hydrogen ratio of the methyl site (D/H_I) with latitude was confirmed but this was not the case for the D/H ratio of the methylene site (D/H_II) and the parameter R [2(D/H_II)/(D/H_I)] did not add any additional information. The frequency distribution of the NMR parameters follows a regular North-South gradient but they are not sufficiently different in each region to allow unique characterisation. The lack of effectiveness of the method, when applied to identification, depends on interannual and interregional variability. As a consequence, all but some very different regions overlap. The SNIF-NMR method is particularly suitable for detecting simple adulterations but, in the Italian situation, seems not to be so useful for solving problems of geographical origin. For this, it could be helpful to use other isotopic parameters and to ameliorate the sampling technique, to improve the effectiveness of the method.

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Regionale und jährliche Variabilität des Ethanolgebundenen Deuteriumgehaltes italienischer Weine

Zusammenfassung Die SNIF-NMR²H-Analytik wird derzeit als Referenzmethode zur Bestimmung der Herkunft des Ethanols bei Produkten aus Weintrauben erfolgreich angewandt. Die Methode wird auch zur Ermittlung der geographischen Herkunft von Wein eingesetzt. Ziel der vorliegenden Arbeit war es, die Brauchbarkeit dieser Analysenmethode zur Bestimmung des geographischen Ursprungs italienischer Weine zu überprüfen. Dazu wurden Analysenergebnisse von 3309 Weinen aus allen 20 Weinbaugebieten Italiens im Rahmen einer Datenbank des D/H-Verhältnisses gesammelt. Während für die D/H_I-Werte eine deutliche Abhängigkeit von der geographischen Breite bestätigt werden konnte, zeigten die D/H_II-Werte keinen derartigen Zusammenhang. Auch der Parameter R erbrachte keine weiteren Informationen. Die Häufigkeits-Verteilung der NMR-Werte folgt deutlich einem Nord-Süd-Gradienten, aber sie sind nicht in jeder Region so unterschiedlich, daß eine signifikante Differenzierung möglich ist. Dies resultiert aus den jahrgangs- und gebietsabhängigen Schwankungen. Die SNIF-NMR Methode ist zur Aufklärung bestimmter Verfälschungen (Zuckerzusatz) gut geeignet, bei italienischen Weinen ist jedoch mit dieser Methode das Problem der Bestimmung der geographischen Herkunft nicht lösbar. Durch Messung weiterer stabiler Isotopen sowie durch eine Verbesserung der Probennahme kann eine Erhöhung der Effizienz zum Nachweis der geographischen Herkunft erzielt werden.

     

Technological characterization of bacteriocin producing Lactococcus lactis strains employed to control Listeria monocytogenes in cottage cheese

In recent years, there has been a particular focus on the application of antimicrobial compounds produced by lactic acid bacteria (LAB) as natural preservatives to control the growth of spoilage and pathogenic bacteria in food. Bacteriocins are antimicrobial peptides which can be added to foods in concentrated forms as food preservatives, e.g. additives, or they can be produced in situ by starters or protective cultures. In this study, twenty Lactococcus lactis bacteriocin producers previously isolated from Italian fermented foods were subjected to a variety of physical and biochemical tests in order to identify those with the greatest potential as starter cultures in cheese production. Of these, four strains isolated from cheese (one nisin Z producer, one nisin A producer and two lacticin 481 producers) which fulfilled the desired technological criteria were assessed for their ability to control Listeria monocytogenes. The subsequent application of these bacteriocinogenic strains as starter cultures in Cottage cheese established that the nisin A producing Lact. lactis 40FEL3, and to a lesser extent the lacticin 481 producers 32FL1 and 32FL3, successfully controlled the growth of the pathogen. This is the first study to directly compare the ability of nisin A, nisin Z and lacticin 481 producing strains to control listerial growth during the manufacture and storage of Cottage cheese.     

Temperature and storage effects on antioxidant activity of juice from red and white grapes

The effect of temperature and storage time on the phenolic composition and on the antioxidant activity of grape (Vitis vinifera L.) juices made using a red (Sangiovese, SG) and a white (Muscat of Alexandria, MA) variety was studied. Total phenolics, flavonoids, flavan‐3‐ols and hydroxycinnamoyl‐tartrates (HCT) were determined on grape extracts (GE) and juices. Total anthocyanins and anthocyanins composition were measured on GE. The antioxidant activity was assessed by means of two different in vitro tests: scavenging of the stable 2,2‐diphenyl‐1‐picrylhydrazyl radical and the inhibition of tyrosine nitration mediated by authentic peroxynitrite (ONOO^?). All the juices were analysed after 24 h and 2 weeks of storage by means of two‐way anova (factors being cultivar and temperature). Anthocyanins were not detected in MA extract, whereas in SG their content was 534 mg malvidin‐equivalent per 100 g of dry matter (d.m.) and malvidin derivatives (glycosylated and coumaroylated) were the most represented (respectively 169 and 41 malvidin‐equivalent per 100 g of d.m.). HCT content of the extracts was higher in SG (overall +33%). Also in grape juices, HCT were lower in MA and in this variety, the trans‐fertaric and cis‐coutaric acids were also undetectable. Cultivar effect proved to be highly significant, while no significant differences in the phenolic composition were observed for storage temperatures (4 and ?20 °C) and cultivar × temperature interaction. However, when statistical analysis was focused on each cultivar, MA was found to be more sensible to storage conditions and a significant reduction in total phenolics (?20%) and flavonoids (?53%) content and in the ONOO^? scavenging potential (?32%) was evident after 2 weeks at 4 °C (when compared with the same storage temperature after 24 h). On the contrary, SG juices did not show significant differences among the four storage treatments investigated. These results could be explained suggesting that anthocyanins presence in red grape plays a key role in juice stability.     

Effect of sourdough fermentation on stabilisation,and chemical and nutritional characteristics of wheat germ

Lactic acid bacteria strains were identified from wheat germ by 16S rRNA partial sequencing, subjected to RAPD-PCR typing and screened. Lactobacillus plantarum LB1 and Lactobacillus rossiae LB5 were used as starters to produce sourdough fermented wheat germ (SFWG). The chemical and nutritional characteristics of SFWG were compared to those of the raw wheat germ (RWG). Lipase activity in SFWG was ca. 2.6-fold lower than that found in RWG. As shown by SPME/GC/MS analysis, most of the volatile compounds derived from lipid oxidation during storage (40 days) were at markedly lower levels in SFWG compared to RWG. Fermentation of wheat germ increased of ca. 50% the concentration of free amino acids. Glu markedly decreased in SFWG, due to its conversion in GABA. The concentration of the anti-nutritional factor raffinose also decreased in SFWG. The in vitro protein digestibility, the concentration of total phenols, phytase and antioxidant activities were increased by fermentation.     

Juice of New citrus hybrids (Citrus clementina Hort. ex Tan.×C. sinensis L. Osbeck) as a source of natural antioxidants

Fruit juices of pigmented and non-pigmented new citrus hybrids obtained by crossing clementine cv. Oroval with different cultivars of blood oranges were analysed to determine parameters related to fruit quality (total soluble solids titratable acidity, pH), and the content of ascorbic acid, total phenolics, flavanones, anthocyanins and phenolic acids. Antioxidant properties were evaluated by oxygen radical absorbance capacity (ORAC), and inhibition of induced linoleic acid peroxidation (InLAP) assays. The results of this study show that some hybrids with high antioxidant activity owing to considerable polyphenol content may be considered rich sources of phytochemicals. The OTA 9 hybrid was shown to be richest in polyphenols, suggesting that consumption of OTA 9 fruit or juice could be useful in health promotion and a disease-preventing diet. Moreover, the juice of this hybrid could be used as raw material to produce antioxidant ingredients for dietary, pharmaceutical, or cosmetic purposes.     

Lactic acid bacteria ecology of three traditional fermented sausages produced in the North of Italy as determined by molecular methods

In this study the bacterial biodiversity during the maturation process of three traditional sausages produced in the North of Italy (Salame bergamasco, Salame cremonese and Salame mantovano) was investigated by using culture-dependent and -independent methods. Eleven plants, in the three provinces considered here, were selected because starter cultures were never used in the production. The bacterial ecology, as determined by plate counts, was dominated by lactic acid bacteria (LAB), with minor contribution of coagulase negative cocci and yeasts. After molecular identification of 486 LAB strains, the species more frequently isolated were Lactobacillus sakei and Lactobacillus curvatus. This evidence was also confirmed by PCR-Denaturing Gradient Gel Electrophoresis (DGGE). All the samples analyzed were characterized by the constant presence of L. sakei and L. curvatus bands. A richer biodiversity was only detected at the beginning of maturation. The results obtained by the molecular characterization of the L. sakei and L. curvatus and by the cluster analysis of the DGGE profiles highlighted a plant-specific population, rather than a geographic characterization of the products, underlining how the environmental and processing conditions are able to select specific microbiota responsible for the main transformations during the fermentation and ripening of the sausages.     

Description of the microflora of sourdoughs by culture-dependent and culture-independent methods

Four types of sourdoughs (L, C, B, Q) from artisanal bakeries in Northern Italy were studied using culture-dependent and culture-independent methods. In all samples, the yeast numbers ranged from 160 to 10⁷ cfu/g, and the numbers of lactic acid bacteria (LAB) ranged from 10³ to 10⁹ cfu/g. The isolated LAB were sequenced, and a similarity was noted between two samples (C, Q), both in terms of the species that were present and in terms of the percentage of isolates. In these two samples, Lactobacillus plantarum accounted for 73% and 89% of the bacteria, and Lactobacillus brevis represented 27% and 11%. In the third sample (B), however, the dominant LAB isolate was Lb. brevis (73%), while Lb. plantarum accounted for only 27%. The fourth sourdough (L) was completely different from the others. In this sample, the most prominent isolate was Weisella cibaria (56%), followed by Lb. plantarum (36%) and Pediococcus pentosaceus (8%). In three out of four samples (L, C and Q), all of the yeasts isolated were identified as Saccharomyces cerevisiae, yet only Candida humilis (90%) and Candida milleri (10%) were isolated in the fourth sample (B). The microbial ecology of the sourdoughs was also examined with direct methods. The results obtained by culture-independent methods and DGGE analysis underline a partial correspondence between the DNA and RNA analysis. These results demonstrate the importance of using a combined analytical approach to explore the microbial communities of sourdoughs.     

Validation of a 1-Day Analytical Diagnostic Real-Time PCR for the Detection of Salmonella in Different Food Meat Categories

Foodborne disease caused by Salmonella represents a worldwide public health problem. In Europe, salmonellosis is still the second most commonly recorded zoonosis. Since the standard culture method for detecting Salmonella (ISO 6579:2002) requires up to 5 days to produce results, the need to develop rapid methods represents an important issue for the authorities and the producers. The aim of the present study was the in-house validation, according to ISO 16140, of an open-formula diagnostic real-time PCR for the detection of Salmonella in all the different meat categories reported in the EU Regulations relative to microbiological criteria for food safety. The assay employed specific primers and a probe target within the ttrRSBCA locus, which allows the tetrathionate respiration in Salmonella. Selectivity, relative accuracy, relative sensitivity and relative specificity were established by testing 110 bacterial strains and 175 various edible meat samples. Results showed 100 % selectivity, 100 % relative accuracy, 100 % relative sensitivity and 100 % relative specificity of the real-time PCR when compared to the standard culture method used as reference. In addition, in order to minimize the effect of the competitive micro-flora naturally present on meat samples, a highly nutritious and selective commercial medium (ONE Broth Salmonella, Oxoid) was evaluated in comparison with the classical non-selective pre-enrichment broth (buffered peptone water). Results demonstrated that the ONE Broth Salmonella medium increases the growth of Salmonella in the presence of competitive micro-flora.     

Evaluation of Pseudomonas spp. through O2 and CO2 headspace analysis

This article proposes an approach to determine the level of Pseudomonas spp. in milk, based on the evaluation of the content of oxygen and carbon dioxide produced in the headspace of sealed vials; the research was divided into two phases: model building and preliminary validation. Three different strains of Pseudomonas spp., Ps. putida (wild strain) and Ps. fluorescens (wild and collection isolates), were used as targets. Data of CO₂ and O₂ were modelled through a modified positive (CO₂) or a negative Gompertz equation (O₂) to estimate the Minimum Detection Time (MDT), defined as the time to attain 3% of CO₂ (MDT₁) or a decrease in the content of O₂ by 3% (MDT₂). Then, MDT₁ and MDT₂ were submitted to a linear regression procedure, using cell concentration as independent variable; the correlations ‘MDT₁/cell concentration’ and ‘MDT₂/cell concentration’ showed high determination coefficients (>0.983). Moreover, the regression procedure pointed out that both MDT₁ and MDT₂ decreased by ca. 3 h for an increase in cell count of 1 log cfu mL^?1. Preliminary validation in milk pointed out that the error associated with the regression line ‘MDT₂/cell concentration’ was below 5%.