In the present work we developed a MPN quantitative real-time PCR (MPN-qPCR) method for a fast and reliable detection and quantification of Listeria monocytogenes and Escherichia coli O157:H7 in minimally processed vegetables. In order to validate the proposed technique, the results were compared with conventional MPN followed by phenotypic and biochemical assays methods. When L. monocytogenes and E. coli O157:H7 were artificially inoculated in fresh-cut vegetables, a concentration as low as 1 CFU g−1 couldbe detected in48hours for bothpathogens. qPCRalone allowed a limit of detectionof 101 CFU g−1 after 2 hours of enrichment for L. monocytogenes and E. coli O157:H7. Since minimally processed ready-to-eat vegetables are characterized by very short shelf life, ourmethod can potentially address the consistent reduction of time for microbial analysis, allowing a better management of quality control. Moreover, the occurrences of both pathogenic bacteria in mixed salad samples and fresh-cut melons were monitored in two production plants from the receipt of the rawmaterials to the early stages of shelf life. No sample was found to be contaminated by L. monocytogenes. One sample of raw mixed salad was found positive to an H7 enterohemorrhagic serotype.

Oenococcus oeni is the main lactic acid bacterium that carries out the malolactic fermentation in virtually all red wines and in some white and sparkling wines. Oenococcus oeni possesses an array of metabolic activities that can modify the taste and aromatic properties of wine. There is, therefore, industrial interest in the proteins involved in these metabolic pathways and related transport systems of this bacterium. In this work, we report the characterization of the O. oeni ATCC BAA-1163 proteome. Total and membrane protein preparations from O. oeni were standardized and analysed by two-dimensional gel electrophoresis. Using tandem mass spectrometry, we identified 224 different spots corresponding to 152 unique proteins, which have been classified by their putative function and subjected to bioinformatics analysis.

The aim of the present study was to establish a new procedure for the oenological selection of Saccharomyces cerevisiae strains isolated from natural must fermentations of an important Italian grape cultivar, denoted as “Negroamaro”. For this purpose, 108 S. cerevisiae strains were selected as they did not produce H2S and then assayed by microfermentation tests. The adopted procedure made it possible to identify 10 strains that were low producers of acetic acid and hydrogen sulphide and showed that they completed sugar consumption during fermentation. These strains were characterized for their speciWc oenological and technological properties and, two of them, strains 6993 and 6920, are good candidates as industrial starter cultures. A novel protocol was set up for their biomass production and they were employed for industrial-scale fermentation in two industrial cellars. The two strains successfully dominated the fermentation process and contributed to increasing the wines’ organoleptic quality. The proposed procedure could be very eVective for selecting “companyspeciWc” yeast strains, ideal for the production of typical regional wines. “Winery” starter cultures could be produced on request in a small plant just before or during the vintage season and distributed as a fresh liquid concentrate culture.

Aims To characterize Oenococcus oeni strains isolated from North-Apulian wines where malic acid degradation is usually achieved by spontaneous fermentations, and to determine the influence of bacterial inoculation time on the malolactic performances in ‘Nero di Troia’ wine using a complete autochthonous microbial regime. Methods and Results: O. oeni strains from wines produced with the autochthonous (Apulia Region, southern Italy) grape variety 'Uva di Troia' were isolated, selected and characterized. MLST and VNTR analysis were used to investigate intraspecific diversity. O. oeni strains were tested in co-inoculation and in sequential inoculation, with two autochthonous yeast strains previously isolated from ‘Nero di Troia’ wine. After a preliminary screening using co-inoculation regime, the O. oeni strains were grouped in reason of the different behavior in malic acid performances. Results suggested that the efficient degradation of malic acid in co-inoculation is a strain-dependent characteristic. Conclusions Autochthonous yeast/bacterium combinations were identified as starter culture, and used in a co-inoculation approach, for vinification of regional wines. Significance and Impact of Study The ‘microbial terroir’ of typical fermented food and beverage production represents a dynamic sector of applied research in food microbiology. In this work we propose the use of autochthonous bacteria and yeast for wine production from an indigenous grape variety.

Currently, the majority of prebiotics in the market are derived from non-digestible oligosaccharides. Very few studies have focused on non-digestible long chain complex polysaccharides in relation to their potential as novel prebiotics. Cereals β-glucans have been investigated for immune-modulating properties and beneficial effects on obesity, cardiovascular diseases, diabetes, and cholesterol levels. Moreover, β-glucans have been reported to be highly fermentable by the intestinal microbiota in the caecum and colon, and can enhance both growth rate and lactic acid production of microbes isolated from the human intestine. In this work, we report the effects of food matrices containing barley β-glucans on growth and probiotic features of four Lactobacillus strains. Such matrices were able to improve the growth rate of the tested bacteria both in unstressed conditions and, importantly, after exposure to in vitro simulation of the digestive tract. Moreover, the effect of β-glucans-containing food on bacterial adhesion to enterocyte-like cells was analyzed and a positive influence on probiotic-enterocyte interaction was observed.

Probiotics, prebiotics and synbiotics are today frequently used components for the elaboration of functional food. Currently, most of the commercialized probiotics are limited to a few strains of the genera Bifidobacteria, Lactobacillus and Streptococcus, most of which produce exopolysaccharides (EPS). This suggests that the beneficial properties of these microorganisms may be related to the biological activities of these biopolymers. In this work we report that a 2-substituted-(1,3)-β-D-glucan of non-dairy bacterial origin has aprebiotic effect on three probiotic strains. Moreover, the presence of this β-D-glucan potentiates in vitro adhesion of the probiotic Lactobacillus plantarum WCFS1 to human intestinal epithelial cells.

This work is the first large-scale study on vineyard-associated yeast strains from Apulia (Southern Italy). Yeasts were identified by Internal Transcribed Spacer (ITS) ribotyping and bioinformatic analysis. The polymorphism of interdelta elements was used to differentiate Saccharomyces cerevisiae strains. Twenty different species belonging to 9 genera were identified. Predominant on the grape surface were Metschnikowia pulcherrima, Hanseniaspora uvarum and Aureobasidium pullulans, whereas M. pulcherrima and H. uvarum were dominant in the early fermentation stage. A total of 692 S. cerevisiae isolates were identified and a number of S. cerevisiae strains, ranging from 26 to 55, was detected in each of the eight fermentations. The strains were tested for biogenic amines (BAs) production, either in synthetic media or grape must. Two Pichia manshurica, an Issatchenkia terricola and a M. pulcherrima strains were able to produce histamine and cadaverine, during must fermentation. The production of BAs in wine must was different than that observed in the synthetic medium. This feature indicate the importance of an “in grape must” assessment of BAs producing yeast. Overall, our results suggest the importance of microbiological control during wine-making to reduce the potential health risk for consumer represented by these spoilage yeasts.

Biogenic amines in wine represent a toxicological risk for the health of the consumer, with several trade implications. In this study 26 strains of Lactobacillus plantarum were analysed for their ability to degrade biogenic amines commonly found during wine fermentation. Two strains of L. plantarum were selected in reason of their ability to degrade putrescine and tyramine. The degradation was assessed in vitro, both in presence of the biogenic amines and in presence of the specific chemical precursor and of producer bacteria. The two L. plantarum biotypes were found capable to work synergically. In addition, the survival in wine-like medium and the aptitude to degrade malic acid after alcoholic fermentation of the selected L. plantarum strains was analysed. Our results suggest the potential application of wine L. plantarum strains to design malolactic starter cultures able to degrade biogenic amines in wine.

Malolactic fermentation (MLF), the bacterial conversion of L-malic into L-lactic acid and carbon dioxide, is encouraged, especially for red wines, to eliminate the taste of malic acid, to decrease the acidity of the wine and to assure the biological stability of wines. However, indigenous lactic acid bacteria (LAB) can start spontaneous MLF after the alcoholic fermentation (AF), affecting negatively wine quality. A controlled MLF can be achieved with inoculation of selected LAB. Oenococcus oeni is the LAB that occurs naturally in wine. It is the dominant bacterial species found during the MLF, being well adapted to the harsh wine conditions, and it possesses the finest oenological malolactic characteristics. The key criteria to select O. oeni strains suitable for MLF are mainly related to (i) the ability to tolerate the harsh conditions encountered during wine fermentation (e.g., low pH, high ethanol concentration and sulphites tolerance, resistance to specific bacteriophages), (ii) physiological and biochemical properties (e.g., high malolactic activity, production of volatile compounds, growth rate, interaction with yeasts responsible of AF) and (iii) technological (e.g., resistance to freeze-drying) properties. Usually, oenological proprieties of O. oeni are strain-specific and this feature might influence differently the aromatic compounds of wine. In this work, a linkeage between stress markers and technological properties of indigenous O. oeni strains isolated from red wines deriving from “Nero di Troia” grapes, an autochthonous Apulian grape variety is reported. The Apulian wine production is characterized by quantity rather than quality, thus the improvement of MLF performances represents one of the key levers to reverse this negative tendence.

Ingestion of fermented foods containing high levels of biogenic amines (BA) can be deleterious to human health. Less obvious is the threat posed by BA producing organisms contained within the food which, in principle, could form BA after ingestion even if the food product itself does not initially contain high BA levels. In this work we have investigated the production of tyramine and putrescine by Lactobacillus brevis IOEB 9809, of wine origin, under simulated gastrointestinal tract (GIT) conditions. An in vitro model that simulates the normal physiological conditions in the human digestive tract, as well as Caco-2 epithelial human cell lines, were used to challenge L. brevis IOEB 9809, which produced both tyramine and putrescine under all conditions tested. In the presence of BA precursors and under mild gastric stress, a correlation between enhancement of bacterial survival and a synchronous transcriptional activation of the tyramine and putrescine biosynthetic pathways was detected. High levels of both BA were observed after exposure of the bacterium to Caco-2 cells. L. brevis IOEB 9809 can produce tyrosine and putrescine under simulated human digestive tract conditions. The results indicate that BA production may be a mechanism that increases bacterial survival under gastric stress.

Lactic Acid Bacteria (LAB) were obtained from durum wheat flour samples and screened for roseoflavin-resistant variants in order to isolate natural riboflavin-overproducing strains. Two riboflavin-overproducing strains of Lactobacillus plantarum isolated as described above were used for the preparation of bread (by means of sourdough fermentation) and pasta (using a pre fermentation step) in order to enhance their vitamin B2 content. Pasta was produced from a monovarietal semolina obtained from the durum wheat cultivar PR22D89 and from a commercial re-milled semolina. Several samples were collected during the pasta-making process (dough, extruded, dried and and cooked pasta) and tested for their riboflavine content by a high performance liquid chromatography method. The applied approaches resulted in a considerable increase of vitamin B2 content (about a two-fold and three-fold increase in pasta and bread, respectively), thus representing a convenient and efficient food-grade biotechnological application for the production of vitamin B2-enriched bread and pasta. This methodology may be extended to a wide range of cereal-based foods, feed and beverages. Additionally, our work exemplifies the production of a functional food by a novel biotechnological exploitation of LAB in pasta-making.

Cereals-derived foods represent a key constituent in the diet of many populations. In particular, pasta is consumed in large quantities throughout the world in reason of its nutritive importance, containing significant amounts of complex carbohydrates, proteins, B-vitamins, and iron. Lactic acid bacteria (LAB) are a heterogeneous group of bacteria that play a key role in the production of fermented foods and beverages with high relevance for human and animal health. A wide literature testifies the multifaceted importance of LAB biotechnological applications in cereal-based products. Several studies focused on LAB isolation and characterization in durum wheat environment, in some cases with preliminary experimental applications of LAB in pasta-making. In this paper, using sourdough as a model, we focus on the relevant state-of-art to introduce a LAB-based biotechnological step in industrial pasta-making, a potential world driver of innovation that might represent a cutting-edge advancement in pasta production.

FAO and WHO encourage studies that implement in vitro experimental protocols to pre-screen novel probiotics and/or attest the efficacy of claimed probiotic strains [1]. To be effective, orally-delivered probiotics should reach the human intestine in relatively high numbers and in a viable state; therefore, they have to tolerate the stresses associated to the gastro-intestinal (GI) environment. All along the different GI sections, bacteria are challenged by several sources of stress, including the action of digestive enzymes, low pH and emulsifying bile salts. Bacterial cells are naturally equipped with various defense mechanisms to enhance survival in hostile environments [2]; these include chaperones to assist the re-folding of denatured proteins and proteases which degrade irreversibly damaged proteins. The food matrix used to deliver microbes may considerably contribute to their probiotic action, e.g. by enhancing survival to stress, stimulating selective growth and favouring gut colonization. Lactobacillus plantarum (Lp) is a widespread lactic acid bacterium which is recognized as a probiotic and is traditionally employed as a food starter. The stress tolerance of Lp WCFS1 was examined in a previously developed in vitro system that simulates the human GI tract [3]. Different carrier matrices were used to assess their protective and buffering properties. Higher survival was observed for bacteria included in complex and/or nutrient-rich matrices, and when potential prebiotics were added, thus highlighting the relevance of matrix composition in shielding and growth-promoting effects. The molecular response of Lp WCFS1 to the simulated GI system was analysed by studying the transcriptional pattern of bacterial genes involved both in stress response (i.e. chaperones, shsp, proteases) and in exerting beneficial effects on the host (i.e. bacteriocins, adhesion factors). Transcription of bacterial stress-related genes remarkably matched the extent of stress during the GI transit, as revealed by the observed survival rate. Indeed, the GI steps of higher mortality corresponded to major induction of typical stress genes, thus implying their involvement in the mechanisms of cellular adaptation to GI stress. GI environment consistently up-regulated probiosis-associated genes. Data obtained with the different vehicle matrices may be valuable for the development of protective and host/bacteria-friendly carriers in the design of functional food. Transcriptional analysis paves the way to the use of some bacterial genes as molecular markers for the screening of strains with potential probiotic applications. Our results also suggest cues to improve reliability and performance of in vitro GI simulators. References 1. FAO/WHO (2002) ftp://ftp.fao.org/es/esn/food/wgreport2.pdf. 2. van de Guchte M. et al. (2002) Antonie Van Leeuwenhoek 82, 187 - 216. 3. Bove P. et al. (2012) Appl. Microb. Biotechnol. DOI 10.1007/s00253-012-4031-2.

Many microorganisms are used as starter cultures in several fermented foods and beverages. In general, the choice of starter cultures is fundamental in order to guarantee the quality of final products. For this reason, the inability to form Biogenic Amine (BA) should be an important criteria in the selection of starter cultures for the management of fermented food and beverages. Inoculation with starter cultures that are unable to produce biogenic amines is a viable option for the control of these compounds in wine (Spano et al., 2010). Malolactic fermentation (MLF) of wine generally starts spontaneously when the population of indigenous Lactic Acid Bacteria (LAB) reaches a sufficient level. When the conditions of wine are favourable to the development of BA-producing LAB, spontaneous MLF can lead to the accumulation of significant amounts of BA (Lonvaud-Funel, 2001). In contrast, when MLF is performed under controlled conditions after inoculating the wine with a selected strain of Oenococcus oeni unable to form BA, the amounts of BA are markedly reduced (Martin-Alvarez et al. 2006). It seems that co-inoculation of Saccharomyces cereviseae and Oenococcus oeni starter cultures has the potential to curb BA formation even more than conventional inoculation for malolactic fermentation after the completion of alcoholic fermentation. The present work present the application of realible Saccharomyces cereviseae/Oenococcus oeni association starter cultures in order to reduce BA content (mainly putrescine) in regional wine.

Among Gram-positive bacteria, CtsR (Class Three Stress gene Repressor) mainly regulates the expression of genes encoding the Clp ATPases and the ClpP protease. To gain a better understanding of the biological significance of the CtsR regulon in response to heat-shock conditions, we performed a global proteomic analysis of Lactobacillus plantarum WCFS1 and ΔctsR mutant strains under optimal or heat stress temperatures. Total protein extracts from bacterial cells were analyzed by two-dimensional gel fractionation. By comparing maps from different culture conditions and different L. plantarum strains, image analysis revealed 23 spots with altered levels of expression. The proteomic analysis of L. plantarum WCFS1 and ctsR mutant strains confirms at the translational level the CtsR-mediated regulation of some members of the Clp family, as well as the heat induction of typical stress response genes. Heat activation of the putative CtsR regulon genes at transcriptional and translational levels, in the ΔctsR mutant, suggests additional regulative mechanisms, as is the case of hsp1. Furthermore, isoforms of ClpE with different molecular mass were found, which might contribute to CtsR quality control.

In this chapter, biogenic amine (BA) production in dairy products, beverages and sausages is described, and their potential harm to human health is addressed. To control the production of toxic compounds it is necessary to know how they are produced, and under which environmental conditions their synthesis is induced. Therefore, the molecular bases for BA production are discussed here. Techniques for detection of the BAs as well as their producing organisms are also described in this chapter, as are current and future strategies to control production. Finally, legislation relating to BA content in food is presented.

Listeria monocytogenes is a gram positive, rod shaped, pathogenic bacterium, causative agent of a severe infection generally known as listeriosis. Packaging and storage conditions of fresh cut vegetables may favour the growth of this psychrotrophic pathogen leading to potential health threat. Detection and enumeration of L. monocytogenes in concentrations up to 103 CFU/g, usually implies use of the most-probable-number technique (MPN) which may take up to seven days for verified identification of the pathogen. We developed a fast and reliable protocol combining MPN with a Real-Time quantitative PCR (qPCR) approach. Samples of fresh cut salads (25 g) purchased at local shops were spiked with 1 to 105 CFU/g of L. monocytogenes. Samples were homogenized, and triplicate series of tubes containing 10-5 to 10 g of food were incubated in Fraser broth at 30 °C for 48 h for standard MPN analysis. After incubation, broth samples were taken from each tube and DNA was extracted. DNA from enrichment tubes was used as template in a qPCR assay targeting a 64 bp hlyA gene sequence of L. monocytogenes. Results of this assay were than compared with those of standard MPN analysis and a complete accordance was observed. Furthermore, we tested an enrichment free approach using the same qPCR assay. Samples were prepared as described for MPN-qPCR while DNA extraction was performed prior to enrichment of inoculated salads. This approach allowed us to identify L. monocytogenes in samples spiked with 10-105 CFU/g. The whole process, including DNA extraction, required less than four hours, thus providing a fast and reliable tool for detection of L. monocytogenes in fresh cut vegetables.

Biogenic amines are small-molecular-weight organic bases that can be encountered in all fermented foods, including wine. Ingestion of wine containing biogenic amines, and especially histamine, can result in health nuisances. HPLC is the analytical technique most often employed in the determination of biogenic amines in wine but HPLC-based methods are expensive and time-consuming. A new method, based upon amine dansylation and TLC/densitometry, was developed and validated. This allowed for the determination of histamine, tyramine, putrescine and cadaverine in wine at concentrations between 1 and 20 mg/L. Analytical performances adequately complied with the needs of routine wine analysis, moreover the method was high-throughput and inexpensive. A simpler, semi quantitative version of the method, based on visual evaluation of spot intensity, was also developed.

Bacillus coagulans GBI-30, 6086 is a safe strain, already available on the market, and characterized by certified beneficial effects. The draft genome sequence presented here constitutes the first pillar toward the identification of the molecular mechanisms responsible for its positive features and safety.

Lactobacillus collinoides CUPV237 is a strain isolated from a Basque cider. Lactobacillus collinoides is one of the most frequent species found in cider from Spain, France, or England. A notable feature of the L. collinoides CUPV237 strain is its ability to produce exopolysaccharides.

Leafy vegetables are consumed fresh after harvest with bland washes, not always efficient in removing pathogenic bacteria that may be attached to the external skin or surfaces. In this work, an optimized ELISA based method developed in our laboratory was used to detect the presence of Listeria monocytogenes in fresh-cut rocket leaves. From a certain quantity (5 g) of fresh rocket leaves contaminated with Listeria monocytogenes, bacteria were isolated and the pathogen was detected using an ELISA protocol. The preliminary results are promising in the use of antibody-antigen interaction to verify the presence of L. monocytogenes in the minimally processed vegetable distribution chain.

Aims: Lactobacillus brevis IOEB 9809 is able to produce both tyramine and putrescine via tyrosine decarboxylase and agmatine deiminase enzymes, respectively, when cultured on synthetic media. The aims of this study were to assess the expression of L. brevis IOEB 9809 tdc and aguA1 genes, during wine fermentation and to evaluate the effect of substrate availability and pH on tdc and aguA1 expression, as well as on biogenic amine production and L. brevis viability. Methods and Results: The relative expression of L. brevis IOEB 9809 tdc and aguA1 genes was analysed in wine by quantitative real-time RT-PCR (qRTPCR) during a period of incubation of 30 days. Cell viability, pH values, putrescine and tyramine concentration were monitored throughout the experiments. Conclusions: The wine trials indicated that L. brevis IOEB 9809 is able to produce both tyramine and putrescine during wine fermentation. Increased cell viability was also observed in wine supplemented with tyrosine or agmatine. qRT-PCR analysis suggests a strong influence of substrate availability on the expression of genes coding for tyrosine decarboxylase and agmatine deiminase in L. brevis IOEB 9809. Less evident is the relationship between putrescine and tyramine production and tolerance to wine pH. Significance and Impact of Study: To our knowledge, this study represents the first assessment of relative expression of L. brevis IOEB 9809 genes involved in biogenic amine production in wine. Furthermore, an effect of biogenic amine production on viability of L. brevis during wine fermentation was established.

Over the past decades, traditional food systems have evolved from poorly coordinated networks to globalized complexes of regulated trade, and the geographical indications (GIs) agro-food market size is approximately $50 billion. Belonging to the intellectual property law as collective property rights, the “GI is a sign used on goods that have a specific geographical origin and possess qualities, reputation, or characteristics that are essentially attributable to that place of origin” (World Intellectual Property Organization (WIPO), 2011). The global impact of “GI” is widely testified through the scientific, social, and economic importance of traditional foods (World Intellectual Property Organization (WIPO), 2011; World Trade Organization (WTO), 2011). In fact, GIs are known to be the earliest type of trademarks. From an edible perspective, this concept is simply and well presented by Bisson et  al. (2002): “consumers expect wine from a particular region to possess unique qualities that differentiate it from other wines of the same varietal from other regions.” The GI system is based upon the concept of “terroir,” a French word used to describe all geographical aspects of the environment, including the climate, geology, cultivar, human, technical, and cultural practices (and the interactions of these factors) that can influence local production.

Due to the increasing interest for healthy foods, the feasibility of using fresh-cut fruits to vehicle probiotic microorganisms is arising scientific interest. With this aim, the survival of probiotic lactic acid bacteria, belonging to Lactobacillus plantarum and Lactobacillus fermentum species, was monitored on artificially inoculated pineapple pieces throughout storage. The main nutritional, physicochemical and sensorial parameters of minimally processed pineapples were monitored. Finally, probiotic Lactobacillus were further investigated for their antagonistic effect against Listeria monocytogenes and Escherichia coli O157:H7 on pineapple pieces. Our results show that at eight-days of storage, the concentration of L. plantarum and L. fermentum on pineapples pieces ranged between 7.3 and 6.3 log cfu g-1 respectively, without affecting the final quality of the fresh-cut pineapple. The antagonistic assays indicated that L. fermentum was able to inhibit the growth of both pathogens, while L. plantarum was effective only against L. monocytogenes. This study suggest that both L. plantarum and L. fermentum could be successfully applied during processing of fresh-cut pineapples, contributing at the same time to inducing a protective effect against relevant foodborne pathogens.

In this study, we investigated the multifunctionality (microbial starters and probiotics) of Lactobacillus plantarum WCFS1 and Lactobacillus plantarum CECT 8328 strains used as microbial starters for the production of yogurt in combination with Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus. The ability of the probiotic strains to survive to oro-gastrointestinal stresses was monitored by an in vitro assay simulating the human digestive tract. The transcriptional level of several genes involved in the immune response suggested that the probiotic strains may have a favorable influence on immunomodulation. Overall, this study revealed that the tested lactobacilli exhibited suitable technological features for yogurt production and might be used to formulate novel food with immunomodulating effects.

Oenococcus oeni is the principal lactic acid bacterium responsible for malolactic fermentation in wine. Here, we announce the genome sequences of five Oenococcus oeni strains isolated from ‘Nero di Troia’ wine undergoing spontaneous malolactic fermentation, reporting, for the first time, several genome sequences of strains isolated from the same terroir.

Oenococcus oeni OM27 is a strain selected from “Nero di Troia” wine undergoing spontaneous malolactic fermentation. “Nero di Troia” is a wine made from “Uva di Troia” grapes, an autochthonous black grape variety from the Apulian region (south of Italy). In this paper we present a 1.78-Mb assembly of the O. oeni OM27 genome, the first fully assembled genome of an O. oeni strain from an Italian wine.

The occurrence of biogenic amines (and especially histamine) in wine constitutes an increasing concern within the wine sector. The analysis of biogenic amines in food is usually carried out by HPLC and capillary electrophoresis. Detection relies upon a derivatization step, which is performed either before or after separation. HPLC remains the reference method for the determination of biogenic amines in wine according to the Organisation Internationale de la Vigne et du Vin. This technique, though precise and sensitive, is expensive and time-consuming, moreover HPLC analysis demands a costly apparatuses and trained personnel. The wine sector would benefit from the development of an alternative analytical technique for the determination of biogenic amines. This should be high-throughput, inexpensive and simple. Thin layer chromatography (TLC) could represent a good alternative to HPLC. TLC methods do not require heavy and/or costly equipment and allow for simultaneous analysis of several samples. The present work presents the development and validation of an analytical method that allows to determine the four main biogenic amines of wine (i.e. histamine, tyramine, putrescine and cadaverine) by means of TLC-densitometry.

A small heat shock gene of Lactobacillus plantarum strain WCFS1 was deleted using a Cre-lox based system. Compared to the wild type, the Δhsp 18.55 mutant strain displayed a similar growth rate when cultivated either under optimal temperature or under different stress conditions such as heat, low pH and salt stress. However, a longer lag phase was observed when the Δhsp 18.55 mutant strain was cultivated under short intense heat stress (50 C). This suggests that the hsp 18.55 gene of L. plantarum may be involved in recovery of L. plantarum stressed cells in the early stage of high temperature stress. In addition, morphology of the mutant cells, investigated by scanning electron microscopy, revealed that cells clumped together and had rough surfaces, and that some of the cells had a shrunken empty appearance, which clearly contrasted with the characteristic rod-shaped, smooth-surface morphology of control L. plantarum cells. Furthermore, inactivation of the hsp 18.55 gene affected membrane fluidity and physicochemical surface properties of L. plantarum WCFS1.

gastric stress, Lactobacillus casei

The yeast Brettanomyces bruxellensis, generally considered the main oenological spoilage microbe, is able to survive during the winemaking process and it confers off-odors to wine, in reason of its ability to produce considerable amounts of volatile phenols. Forty-eight isolates of B. bruxellensis, obtained from several wines collected in Apulia (Southern Italy), were genetically characterized using an integrated approach, including a strain biodiversity analysis by Sau-PCR. Furthermore, the production of volatile phenols was assessed in wine and in synthetic medium, confirming the oenological spoilage potential of the analysed strains. Our findings indicate a remarkable genetic variability of the B. bruxellensis identified strains and corroborate the evidence of a high level of genotypic and phenotypic polymorphism within B. bruxellensis species. Moreover, the observation reported suggest that strains from wines produced in the same geographical areas often clustered differently, indicating a complex intraspecific biodiversity in the regional wine environments. Diversity in volatile phenol production reflects intraspecific biodiversity highlighted by Sau-PCR. Strains diversity linked to differences in ‘spoilage potential’ increase the industrial relevance of this study, allowing the design of new strategies for B. bruxellensis control in wines.

Wheat contains various essential nutrients including the B group of vitamins. However, B group vitamins, normally present in cereals derived products, are easily removed or destroyed during milling, food processing or cooking. Lactic acid bacteria (LAB) are widely used as starter cultures for the fermentation of a large variety of foods and can improve the safety, shelf life, nutritional value, flavor, and overall quality of the fermented products. In this regard, the identification and application of strains delivering health - promoting compounds (nutraceuticals) is a fascinating field. Besides their key role in food fermentations, several LAB are found in the gastrointestinal track (GIT) of humans and animals, are commercially used as probiotics and possess GRAS (Generally Recognized As Safe) status. LAB are usually auxotrophic for several vitamins although certain strains of LAB have the capability to synthesize water-soluble vitamins such as those included in the B group. In recent years, a number of biotechnological processes have been explored to perform a more economical and sustainable vitamin production than that obtained via chemical synthesis. This review article will briefly report the current knowledge on lactic acid bacteria synthesis of vitamins B2, B11 and B12 and the potential strategies to increase B-group vitamin content in cereals-based products, where vitamin producing LAB have been leading to the elaboration of novel fermented functional foods. In addition, the use of genetic strategies to increase vitamin production or to create novel vitamin-producing strains will be also discussed.

The European Food Safety Agency (EFSA) has recently introduced a system for a pre-market safety assessment of selected taxonomic groups of microorganisms leading to a Qualified Presumption of Safety (QPS), European equivalent of the Generally Recognized as Safe (GRAS) status. Several species of food-related lactic acid bacteria (LAB) associated with food, have obtained a QPS status. The adaptability of LAB to fermentation process, their biosynthetic capacity and metabolic versatility, are some of the principal features that facilitate the application of LAB in foods for producing, releasing and/or increasing specific beneficial compounds. Among these, vitamin production by LAB has recently gained attention of the scientific community. The proper selection and exploitation of nutraceutical-producing LAB is an interesting strategy to produce novel fermented foods with increased nutritional and/or health promoting properties. Fermented milks or bread with high levels of B-group vitamins (such as folate and riboflavin) can be produced by LAB-promoted biosynthesis. In this paper, the use of probiotics bacteria such as Lactobacillus plantarum as a strategy for an in situ production and/or overproduction of vitamin B2 will be discussed.

The risk of pathogen contamination and growth is one of the main safety concerns associated with fresh-cut produce, as highlighted by the increasing number of produce-linked foodborne outbreaks in recent years. The pathogens of major concern in freshcut produce are Listeria monocytogenes, pathogenic Escherichia coli, and Salmonella spp. Listeria monocytogenes is able to grow and multiply in vegetables packaged and stored in modified atmosphere. Biofilms formed by Listeria monocytogenes pose a serious threat to the safety of fresh cut produce as they can persist for long periods of time in the food processing environment and thus represent a source of recurrent contamination. Moreover the occurrence of naturally formed biofilms on fresh produce has been demonstrated. In this article the microbiological safety of fresh-cut produce and factors affecting Listeria monocytogenes survival and growth on fresh-cut produce are discussed. Moreover, the structure and physiology of Listeria monocytogenes biofilms are reviewed.

Fermented food Geographical Indications (GIs), protected within the framework of Intellectual Property and Human Rights, are based on various forms of local knowledge and technical practices and on various aspects of local biodiversity, ranging from plant varieties and animal breeds to microbial ecosystem. In the light of the EC Regulation 510/2006 relating to the protection of EU GIs, information about microbial resource management in GIs production is not unequivocally defined. We argue that it is possible to integrate the microbial resource information regimen coherently within the intellectual property framework for GIs. Product specification of GI fermented foods might comprise a list of autochthonous microbial strains representing the “virtuous” microbial biodiversity of a specific terroir and for a given method of food production. The autochthonous strains should be genotypically and technologically characterized and deposited in microbial collections. This approach will be useful in improving the “unique qualities” of products, and in ensuring a strong focus on the biological risks for human health and industrial exigencies of product standardization. Additionally, it is important to underline that a multi-strain starter culture essentially satisfies the patentable criteria.

The development of fast, reliable and culture-independent molecular tools to detect bacteria producing biogenic amines deserves the attention of research and ultimately of the food industry in order to protect consumers’ health. Here we present the application of a simple, low-cost, fast and sensitive method to perform microdroplet-based multiplex PCR, directly on a real food matrix, for the simultaneous detection of bacterial genes involved in biogenic amine biosynthesis. After inoculating wine with Lactobacillus brevis IOEB 9809, cell lysis and DNA amplification are performed in one single step, without preliminary nucleic acid extraction or purification treatments. The assay is performed in about 30 min, requiring 150 nL of starting sample and it enables the detection of down to 15 bacterial cells. With respect to traditional culture techniques, the speed, the simplicity and the cheapness of this procedure allows an effective monitoring of microbial cells during food-making and processing.

Here we describe the draft genome sequence and annotation of Lactobacillus plantarum strain Lp90, the first sequenced genome of a L. plantarum strain isolated from wine. This strain has a noticeable ropy phenotype and showed potential probiotic properties. The genome consists of 3,324,076 bp (33 contigs) and contains 3,155 protein coding genes, 34 pseudo-genes and 84 RNA genes.

The aim of this work was to study the biodiversity of cultivable non-Saccharomyces yeasts isolated from North-Apulian region on the autochthonous wiine variety ‘Uva di Troia’ during vintages 2012 and 2013. Grapes were collected in vineyards from four different geographical areas on which four different wines with the status of geographical indication are produced. Different restriction profiles of ITS–5.8S rDNA region, corresponding to Candida boidinii, Candida zemplinina, Hanseniaspora guilliermondii, Issatchenkia terricola, Zygosaccharomyces bailii, Hanseniaspora uvarum, Zygoascus hellenicus and Hanseniaspora opuntiae was observed. The most abundant genera were represented by Hanseniaspora (about 58%, H. guilliermondii 53%, H. uvarum 4% and H. opuntiae 1%) and Candida (about 32%, C. zemplinina 19% and C. boidinii 16%). Significant differences among locations and vintages. This first report on the non-Saccharomyces diversity during the beginning steps of spontaneous alcoholic fermentations from Nero di Troia wines provides the basis for an improved management of non-Saccharomyces in typical Apulian wines important for the development of the local wine industry and to achieve an enhanced standard of safety in the final productions.

Oenococcus oeni is an environmental lactic acid bacteria species encountered particularly in wine, where it achieves the malolactic fermentation. Molecular typing methods have previously revealed that the species is made of several genetic groups of strains, some being specific to certain types of wines, ciders or regions. Here, we describe 36 recently released O. oeni genomes and the phylogenomic analysis of these 36 plus 14 previously reported genomes. We also report three genome sequences of the sister species Oenococcus kitaharae that were used for phylogenomic reconstructions. Phylogenomic and population structure analyses performed by diverse methods revealed that the 50 O. oeni genomes delineate two major groups of 12 and 37 strains, respectively named A and B, plus a putative group C consisting in a single strain. Strains of genetic groups A/B and C were more distantly related than strains from different subspecies of Leuconostoc mesenteroides. In addition, group A strains proved to be predominant in wine and to form subgroups adapted to specific types of wine such as champagne. The orthologs and SNP contents of the diverse genetic groups revealed some of their specific genetic features. Altogether, the results confirmed that the O. oeni species is made of several genetic groups of strains, whose adaptation to different products such as champagne or cider is reflected at the genetic level. They also suggest that ancestral strains were adapted to low-ethanol containing environments such as fruits and that group A strains were naturally selected in a process of domestication to wine.

In this study, the probiotic potential of Lactobacillus plantarum wild-type and derivative mutant strains was investigated. Bacterial survival was evaluated in an in vitro system, simulating the transit along the human oro-gastro-intestinal tract. Interaction with human gut epithelial cells was studied by assessing bacterial adhesive ability to Caco-2 cells and induction of genes involved in innate immunity. L. plantarum strains were resistant to the combined stress at the various steps of the simulated gastrointestinal tract. Major decreases in the viability of L. plantarum cells were observed mainly under drastic acidic conditions (pH≤2.0) of the gastric compartment. Abiotic stresses associated to small intestine poorly affected bacterial viability. All the bacterial strains significantly adhered to Caco-2 cells, with the ΔctsR mutant strain exhibiting the highest adhesion. Induction of immune-related genes resulted higher upon incubation with heat- inactivated bacteria rather than with live ones. For specific genes, a differential transcriptional pattern was observed upon stimulation with different L. plantarum strains, evidencing a possible role of the knocked out bacterial genes in the modulation of host cell response. In particular, cells from Δhsp18.55 and ΔftsH mutants strongly triggered immune defence genes. Our study highlights the relevance of microbial genetic background in host–probiotic interaction and might contribute to identify candidate bacterial genes and molecules involved in probiosis.

The probiotic potential of Lactobacillus plantarum and Lactobacillus fermentum strains, capable of overproducing riboflavin, was investigated. The riboflavin production was quantified in co-cultures of lactobacilli and human intestinal epithelial cells, and the riboflavin overproduction ability was confirmed. When milk and yogurt were used as carrier matrices, L. plantarum and L. fermentum strains displayed a significant ability to survive through simulated gastrointestinal transit. Adhesion was studied on both biotic and abiotic surfaces. Both strains adhered strongly on Caco-2 cells, negatively influenced the adhesion of Escherichia coli O157:H7, and strongly inhibited the growth of three reference pathogenic microbial strains. Resistance to major antibiotics and potential hemolytic activity were assayed. Overall, this study reveals that these Lactobacillus stains are endowed with promising probiotic properties and thus are candidates for the development of novel functional food which would be both enriched in riboflavin and induce additional health benefits, including a potential in situ riboflavin production, once the microorganisms colonize the host intestine.

The aromatic impact of bakery yeast starters is currently receiving considerable attention. The flavor characteristics of the dough and the finished products are usually evaluated by gas chromatography and sensory analysis. The limit of both techniques resides in their low-throughput character. In the present work, proton-transfer-reaction mass spectrometry (PTR-MS), coupled to a time-of-flight mass analyzer, was employed, for the first time, to measure the volatile fractions of dough and bread, and to monitor Saccharomyces cerevisiae volatile production in a fermented food matrix. Leavening was performed on small-scale (1g) dough samples inoculated with different commercial yeast strains. The leavened doughs were then baked, and volatile profiles were determined during leavening and after baking. The experimental setup included a multifunctional autosampler, which permitted the follow-up of the leavening process on a small scale with a typical throughput of 500 distinct data points in 16 h. The system allowed to pinpoint differences between starter yeast strains in terms of volatile emission kinetics, with repercussions on the final product (i.e. the corresponding micro-loaves). This work demonstrates the applicability of PTR-MS for the study of volatile organic compound production during bread-making, for the automated and online real-time monitoring of the leavening process, and for the characterization and selection of bakery yeast starters in view of their production of volatile compounds

Analytical tools for the identification and quantification of volatile organic compounds (VOCs) produced by microbial cultures have countless applications in an industrial and research context which are still not fully exploited. The various techniques for VOC analysis generally arise from the application of different scientific and technological philosophies, favoring either sample throughput or chemical information. Proton Transfer Reaction-Mass Spectrometry (PTR-MS) represents a valid compromise between the two aforementioned approaches, providing rapid and direct measurements along with highly informative analytical output. The present paper reviews the main applications of PTR-MS in the microbiological field, comprising food, environmental and medical applications.

Lactobacillus fermentum isolated from sourdough was able to produce riboflavin. Spontaneous roseoflavinresistant mutants were obtained by exposing the wild strain (named L. fermentum PBCC11) to increasing concentrations of roseoflavin. Fifteen spontaneous roseoflavin-resistant mutants were isolated, and the level of vitamin B2 was quantified by HPLC. Seven mutant strains produced concentrations of vitamin B2 higher than 1 mg L−1. Interestingly, three mutants were unable to overproduce riboflavin even though they were able to withstand the selective pressure of roseoflavin. Alignment of the rib leader region of PBCC11 and its derivatives showed only point mutations at two neighboring locations of the RFN element. In particular, the highest riboflavinproducing isolates possess an A to G mutation at position 240, while the lowest riboflavin producer carries a T to A substitution at position 236. No mutations were detected in the derivative strains that did not have an overproducing phenotype. The best riboflavin overproducing strain, named L. fermentum PBCC11.5, and its parental strain were used to fortify bread. The effect of two different periods of fermentation on the riboflavin level was compared. Bread produced using the coinoculum yeast and L. fermentum PBCC11.5 led to an approximately twofold increase of final vitamin B2 content

Lactobacilli are found in diverse environments and are widely applied as probiotic, health-promoting food supplements. Polysaccharides are ubiquitously present on the cell surface of lactobacilli and are considered to contribute to the species- and strain-specific probiotic effects that are typically observed. Two Lactobacillus plantarum strains, SF2A35B and Lp90, have an obvious ropy phenotype, implying high extracellular polysaccharide (EPS) production levels. In this work, we set out to identify the genes involved in EPS production in these L. plantarum strains and to demonstrate their role in EPS production by gene deletion analysis. A model L. plantarum strain, WCFS1, and its previously constructed derivative that produced reduced levels of EPS were included as reference strains. The constructed EPS-reduced derivatives were analyzed for the abundance and sugar compositions of their EPS, revealing cps2-like gene clusters in SF2A35B and Lp90 responsible for major EPS production. Moreover, these mutant strains were tested for phenotypic characteristics that are of relevance for their capacity to interact with the host epithelium in the intestinal tract, including bacterial surface properties as well as survival under the stress conditions encountered in the gastrointestinal tract (acid and bile stress). In addition, the Toll-like receptor 2 (TLR2) signaling and immunomodulatory capacities of the EPS-negative derivatives and their respective wild-type strains were compared, revealing strain-specific impacts of EPS on the immunomodulatory properties. Taken together, these experiments illustrate the importance of EPS in L. plantarum strains as a strain-specific determinant in host interaction.

With this book, Maureen O’Malley aimed to fill the gap between the philosophy of microbes and the philosophy of ‘macrobes’ (a neologism coined by O’Malley and Dupre´ to indicate cellular organisms other than microbes). After a conceptual introduction, the first chapter reports two exemplificative case studies, followed by brief perspec- tives concerning the philosophical content of microbiology and about the philosophy of biology in light of microbiolo- gy. At page 41, the reader will find a bottleneck: from this point up to the conclusions, the author will cover topics in which the consideration of microbiology leads one to radi- cally and/or incrementally rethink philosophy of biology assumptions.

Small heat shock proteins (sHsps) are ubiquitous conserved chaperone-like proteins involved in cellular proteins protection under stressful conditions. In this study, a reverse transcription quantitative PCR (RT-qPCR) was developed and used to quantify the transcript level of a small heat shock gene (shs) in the probiotic bacterium Lactobacillus acidophilus NCFM, under stress conditions such as heat (45 °C and 53 °C), bile (0.3% w/v), hyperosmosis (1 M and 2.5 M NaCl), and low pH value (pH 4). The shs gene of L. acidophilus NCFM was induced by salt, high temperature and acidic stress, while repression was observed upon bile stress. Analysis of the 5' noncoding region of the hsp 16 gene reveals the presence of an inverted repeat (IR) sequence (TTAGCACTC-N9-GAGTGCTAA) homologue to the controlling IR of chaperone expression (CIRCE) elements found in the upstream regulatory region of Gram-positive heat shock operons, suggesting that the hsp16 gene of L. acidophilus might be transcriptionally controlled by HrcA. In addition, the alignment of several small heat shock proteins identified so far in lactic acid bacteria, reveals that the Hsp16 of L. acidophilus exhibit a strong evolutionary relationship with members of the Lactobacillus acidophilus group.

Vegetables fermented foods and beverages are traditional worldwide consumed foodstuffs, since fermentation is one of the most important methods of food preservation still in use for vegetables and fruits. Several species of lactic acid bacteria are involved in the fermentation of vegetable matrices. These microorganisms transform raw materials trough their metabolism conferring specific nutritional and organoleptic properties to the product. The industrial demand of standardization of the product and the process, as well as the need to ensure food safety, require to drive a controlled fermentation. Therefore, selected cultures of lactic acid bacteria are recommended as microbial starter for industrial fermentations. In the last years, an increasing interest has been generated about the beneficial role played by lactic acid bacteria in the gut environment. For this reason, fermented food and beverages from vegetables have been proposed as optimal carriers to deliver probiotic bacteria and for the elaboration of functional foods. In this chapter, we shall provide a background on the key role of lactic acid bacteria in vegetable fermentations, focusing on both traditional and innovative applications for the food industry.

The yeast Candida zemplinina (Starmerella bacillaris) is frequently isolated from grape and wine environments. Its enological use in mixed fermentation with S. cerevisiae has been extensively investigated these last years, and several interesting features, including low ethanol production, fructophily, glycerol and other metabolites production, have been described. In addition, molecular tools allowing the characterization of yeast populations have been developed, both at the inter- and intraspecific levels. However, most of these fingerprinting methods are not compatible with population genetics or ecological studies. In this work, we developed ten microsatellite markers for the C. zemplinina species that were used for the genotyping of 163 strains from nature or various enological regions (28 vineyards/wineries from seven countries). We show that the genetic diversity of C. zemplinina is shaped by geographical localisation. Populations isolated from winemaking environments are quite diverse at the genetic level: neither clonal-like behaviour nor specific genetic signature was associated with the different vineyards/wineries. Altogether, these results suggest that C. zemplinina is not under selective pressure in winemaking environments.

A critical feature of probiotic microorganisms is their ability to colonize the intestine of the host. Although the microbial potential to adhere to the human gut lumen has been investigated in in vitro models, there is still much to discover about their in vivo behaviour. Zebrafish is a vertebrate model that is being widely used to investigate various biological processes shared with humans. In this work, we report on the use of the zebrafish model to investigate the in vivo colonization ability of previously characterized probiotic lactic acid bacteria. Lactobacillus plantarum Lp90, L. plantarum B2 and Lactobacillus fermentum PBCC11.5 were fluorescently tagged by transfer of the pRCR12 plasmid, which encodes the mCherry protein and which was constructed in this work. The recombinant bacteria were used to infect germ-free zebrafish larvae. After removal of bacteria, the colonization ability of the strains was monitored until 3 days post-infection by using a fluorescence stereomicroscope. The results indicated differential adhesion capabilities among the strains. Interestingly, a displacement of bacteria from the medium to the posterior intestinal tract was observed as a function of time that suggested a transient colonization by probiotics. Based on fluorescence observation, L. plantarum strains exhibited a more robust adhesion capability. In conclusion, the use of pRCR12 plasmid for labelling Lactobacillus strains provides a powerful and very efficient tool to monitor the in vivo colonization in zebrafish larvae and to investigate the adhesion ability of probiotic microorganisms