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In this study, we focused on the effect of an enterocin or an Enterococcus faecalisstrain added onto sliced dry-cured ham that was artificially inoculated with Listeria monocytogenesand stored at 7°C. The population of L. monocytogenesand the expression of five genes were monitored throughout the storage period. A persistent and a nonpersistent strain were tested, and both were influenced by the presence of the enterocin; both populations were reduced by more than 2 Log CFU/g after 14 days compared with the control, noninoculated ham. The presence of E. faecalis,a bacteriocin-producing lactic acid bacterium, had a less pronounced effect on the viable counts for both strains. Concerning gene expression, a common trend observed for both strains in the presence of enterocin was the down-regulation of genes tested after 30 min of storage at 7°C. For the remainder of the storage period, the expression fluctuated but was mostly reduced. Similarly, the presence of E. faecalisled to an overall down-regulation of genes. The effect on gene expression of both enterocin and E. faecaliswas more pronounced on the nonpersistent L. monocytogenesstrain. Although the potential of a bacteriocin and a bacteriocin-producing microorganism to control L. monocytogeneswas confirmed, this study highlights that gene expression may be influenced and needs to be evaluated when considering such biopreservation interventions.

Listeria monocytogenescan be introduced into food processing plants via raw material of animal or plant origin and can establish endemic populations through formation of biofilms. Biofilms are a continuous source of contamination for food products, and L. monocytogenescells in biofilms are more resistant to stress and sanitizing agents than are planktonic cells. The use of gas-discharge plasmas may offer a feasible alternative to conventional sanitization methods. Plasmas are a mixture of charged particles, chemically reactive species, and UV radiation and can be used to destroy microorganisms. The purpose of this study was to measure the effectiveness of cold atmospheric pressure plasma (APP) treatments against bacteria attached to a solid surface and to evaluate the individual susceptibility of various L. monocytogenesstrains. Attention was focused on the state of the cells after treatment, combining detection by viable counts and quantitative PCR (qPCR). Most of the culturable cells were inactivated after APP treatment, but the qPCR assay targeting the 16S rRNA revealed the presence of injured cells or their entrance into the viable but nonculturable state. These results were at least partly confirmed by a resuscitation experiment. After APP treatment, L. monocytogenescell suspensions were incubated in brain heart infusion broth; some cells grew in the medium and therefore had survived the treatment. An understanding of the effects of APP on L. monocytogenescan inform the development of sanitation programs incorporating APP for pathogen removal. Methods other than those based of the culturability of the cells should be used to monitor pathogens in food processing plants because cultivation alone may underestimate the actual microbial load.

•Wine fermentations are complex ecosystems in which non-Saccharomycesand Saccharomyces cerevisiaeyeasts are interacting.•Non-Saccharomycespossess interesting physiological characteristics which can be exploited in the winemaking process.•Starmerella bacillarisshow fructophylic, cryotellerant and osmophylic characters of interest for the winemakers.•Starmerella bacillariscan contribute to the reduction of ethanol in table wines and acetic acid in sweet wines.

•Bioinformatics tools have been developed to provide information on microbe diversity.•Shotgun metagenomics is a promising approach to discover the functions of microbiota.•Data generated through a multi-omics approach can improve the knowledge on what happens in food.

Technological properties and biogenic amine production were analyzed in 56 bacteriocinogenic lactococci and enterococci strains isolated from raw goat's milk. Fifteen lactococci strains were able to reduce milk pH to 5.3 or lower after 6 h, while enterococci strains were initially slow in producing acids. Lactococcus lactissubsp. lactisGLc06 and three strains of Enterococcus faecalis(GEn20, GEn22, and GEn23) presented high proteolytic activity. L. lactissubsp. lactisGLc06 and E. faecalisGEn22 also showed a high percentage of autolysis after only 4 h, reaching 71.11 and 97.67%, respectively, after 24 h. No strain was able to secrete exopolysaccharides, and L. lactissubsp. lactisGLc22 and 25 of the Enterococcusstrains were able to produce diacetyl. L. lactissubsp. lactisGLc05 and 23 of the Enterococcusstrains presented a high tolerance to NaCl at 10% (wt/vol). Regarding biogenic amine production, 12 strains (5 lactococci and 7 enterococci) were capable of forming tyramine and 4 strains (1 lactococcus and 3 enterococci) were capable of forming 2-phenylethylamine, but in very low amounts. GLc06 presented great acidifying, proteolytic, and autolytic activities, and GLc05 was capable of growing at high NaCl concentrations (10%, wt/vol), possessing medium autolytic and proteolytic activities. Some enterococci strains produced diacetyl and high autolytic and extracellular proteolytic activities and also presented resistance to high NaCl concentrations. The interesting technological properties presented by some bacteriocinogenic strains can justify their use by the dairy industry, with the aim of ensuring both safety due to bacteriocin production and technological transformations in fermented products.

Table olives (Olea europaeaL.) are one of the most important fermented vegetables worldwide, whereas sea fennel (Crithmum maritimumL.) represents an emerging food crop, characterized by interesting nutritional and sensory qualities. Both are characterized by a high concentration of bioactive compounds with health beneficial effects. Thanks to these features, table olives and sea fennel undoubtedly represent two valuable ingredients for the manufacture of innovative vegetable preserves. Given these premises, the present study was aimed at exploring the co-fermentation of green olives and sea fennel to produce laboratory-scale prototypes of innovative high value preserves. To this end, the effects of two recipes, two standard methods for production of table olives, and two fermentation microbiota (resident or inoculated) were assessed. The prototypes were evaluated for their microbial dynamics as well as for key sensory traits by a panel of trained assessors. During the fermentation, all the prototypes showed a progressive pH reduction. Mesophilic lactobacilli, mesophilic lactococci, and yeasts were the main microbial groups at the end of the fermentation, while Enterobacteriaceae decreased during fermentation. Metataxonomic analysis revealed an evolution of the microbiota, with Lactiplantibacillus plantarumdominating in all the prototypes in the late stage of fermentation, irrespective of the recipe, processing method, and starter inoculation. A greater crunchiness and lower fibrousness were perceived in the Greek style prototypes, which were preferred than Spanish style prototypes by trained panelists.

A Listeria monocytogenessubgenomic array, targeting 54 genes involved in the adhesion, adaptation, intracellular life cycle, invasion, and regulation of the infection cycle was used to investigate the gene expression patterns of acid- and salt-stressed Listeriacells after exposure to conditions similar to those in gastric and pancreatic fluids. Three L. monocytogenesstrains, one laboratory reference strain (EGDe) and two food isolates (wild strain 12 isolated from milk and wild strain 3 isolated from fermented sausage), were used during the studies. Differences in the expressed genes were observed between the gastric and pancreatic treatments and also between the serotypes. Increased transcripts were observed of the genes belonging to the adaptation and regulation group for serotype 4b (strain 12) and to the invasion and regulation group for serotype 1/2a (strain EGDe). Interestingly, no significantly differentially expressed genes were found for serotype 3c (strain 3) in most cases. The genes related to adaptation (serotype 1/2a) and to intracellular life cycle and invasion (serotype 4b) were down-regulated in order to cope with the hostile environment of the gastric and pancreatic fluids. These findings may provide experimental evidence for the dominance of serotypes 1/2a and 4b in clinical cases of listeriosis and for the sporadic occurrence of serotype 3c.

AbstractIn this article, we developed a biomolecular assay specifically designed for the identification of Listeriaat species level. The proposed test was based on (i) a duplex PCR targeting a specific fragment for L. monocytogenes(plcAgene) and a common fragment for all the Listeria(16S rRNA gene) and (ii) a minisequencing of the common fragment to detect diagnostic sites for the differentiation of the other five species: L. innocua, L. welshimeri, L. seeligeri, L. ivanovii, and L. grayi.The specificity of the assay was first tested on a total of 25 certified strains representing 6 Listeriaspecies and 14 non-Listeriastrains as negative control and validated on 124 wild isolates obtained from food samples. The proposed assay provides an appropriate tool for rapid identification of Listeriaat species level and it should be of great benefit to the food industry as well as to regulatory or public health laboratories engaged in establishing the safety of food products and the management of listeriosis.

Coagulase-negative catalase-positive cocci (CNCPC) play a very important role during the fermentation of sausages. In particular, they are involved in the aroma formation of the final product, because they release lipases that are able to free short-chain fatty acids that are contributing to the sensory characteristics of the fermented sausage. Few studies have been undertaken to elucidate the regulation of lipase gene expression in Staphylococcus xylosusby substrate molecules or products of lipolysis. The aim of this study was to analyze the gehMgene expression of S. xylosusDSMZ 6179 in vitro with growth media containing different concentrations of lipids and in situ during the maturation of fermented sausages. The results obtained suggest that a concentration that increases in triglycerides in the growth medium suppresses the expression of the lipase gene.

A quick molecular biology method based on the polymerase chain reaction (PCR) and Denaturing Gradient Gel Electrophoresis (DGGE) was developed for distinguishing strains belonging to the Saccharomycessensu stricto group. Differentiation was obtained between S. cerevisiae, S. paradoxus and S. bayanus/S. pastorianusalthough no distinction was possible between S. bayanusand S. pastorianususing the amplification of the ITS regions. The ability to distinguish between different strains of the Saccharomycessensu stricto group could allow for a better understanding of the ecology of these species on grapes as well as in musts and wines and the method developed can be useful for the quick identification of Saccharomycessensu stricto strains from numerous isolates.

Forty-nine strains of Kloeckera apiculata, isolated from the Friuli region in Italy, were differentiated on the basis of fermentation behaviour and production of secondary compounds in two different grape musts at 18 °C. The isolates exhibited a controlled production of acetic acid, only in a few cases more that 1 g/l. In Moscato grape must the strains exhibited a more uniform behaviour for the production of higher alcohols, ethyl acetate and acetoin than in red grapes. In general, higher levels of ethanol, glycerol and acetic acid were produced in red grape must fermentation. Apiculate strains behaved differently in the two musts, with different metabolic phenotypes dominating the fermentation process. The existence of different metabolic phenotypes correlated with the must composition underlines the need to perform a selection of indigenous apiculate yeasts to obtain the desired consistent products.

A strain of Candida famatawas adapted to high copper concentration (1.26 mM) and a number of biochemical parameters have been tested, in order to get information on the mechanisms of metal toxicity and detoxification as well as on the metabolic responses to the treatment. The cytoplasmic levels of superoxide dismutase, peroxidase and glutathione were found significantly increased with respect to control cells, in contrast to catalase which is not affected. The activities of enolase and of triosephosphate isomerase are found to decrease as a consequence of the exposure to copper. Statistically significant differences in the content of some aminoacids are found between copper-treated and control cells.

In view of the considerable public health risk of the enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli, a multiplex-PCR based method was used for the amplification of the slt genes and of the eaeA gene. Three pairs of primers, different from the oligonucleotides previously used by other authors, were exploited for the amplification. Different E. coli serotypes were tested with the optimized protocol. Fifty three artificially contaminated samples and sixty naturally contaminated samples were processed with the multiplex-PCR. All the artificially contaminated samples gave positive results independently of the number of cells inoculated. On the contrary, the naturally contaminated samples were all negative. The results obtained from this experiment demonstrated that this protocol could be used for monitoring the spread of these organisms in food.

This paper analyses the preferences and emotional connotations of Italian consumers towards different chocolate types and assesses which label information consumers take into consideration during the purchasing process. A survey was conducted to collect data from 390 respondents from two different Italian chocolate production regions (Sicily and Piedmont). The results show that overall, consumers prefer dark, extra-dark and milk chocolate. However, a significantly higher percentage of women and men from Piedmont prefer “Gianduia” (hazelnut-based chocolate), when compared to Sicilians, whereas the type of chocolate formats preferred by consumers differs by gender (P <0.05). Different attitudes before or after chocolate consumption are closely related to a certain type of product, in which gender and the geographical aspect are key influencing factors. Women and men from Piedmont feel unpleasant emotions towards chocolate before and after consumption, this was observed in the correlation analysis where positive correlations between anger emotions before consuming chocolate with guilty and sad emotions after consumption were noted in women and men, respectively (P <0.05). In contrast, no significant correlations between feelings reported before and after chocolate consumption of Sicilian men was observed. During decision making, more than 40% of participants are aware of the cocoa quantity, nutritional information and fair-trade certification on the chocolate label. Our findings provide an empirical basis to inform the chocolate industry regarding consumer attitudes towards chocolate, to raise awareness of the social dimension in food labelling and to provide a baseline for the choice of a marketing communication strategy which plays upon emotional claims.

Strains (107) of L. monocytogeneswere tested with a PCR‐restriction enzyme analysis with two new original primers. A segment of 1395 bp containing the entire iapgene in L. monocytogeneswas amplified by the PCR technique. The PCR product was cleaved with the restriction enzymes HindIII and RsaI, and the fragments generated were separated by gel electrophoresis. Two groups of serovars were obtained: one group contained serovars 1/2a and 1/2c, the other group contained serovars 1/2b, 3b and 4b. The PCR‐restriction enzyme analysis method described in this paper could be a useful tool for the unambiguous division of L. monocytogenesinto two serovar groups, and it could be used to study the evolution of different serotypes and groups of serotypes in foods produced in the same processing plant and processed during the same month. The RE‐PCR method used can give a rapid confirm at the subgroup level in the laboratory of an epidemiological association between human disease and suspected sources of contaminated food.

A polymerase chain reaction based test was developed for the detection of Salmonellaspp. in blood specimens. After amplification of a 389 bp‐polymerase chain reaction product from the invA gene, a microtiter plate hybridization assay was performed. The protocol described allowed the detection of six to seven copies of the Salmonella typhigenome, as determined by serial dilutions of DNA from S. typhi. Eighteen blood specimens from artificially infected rats and 22 blood specimens from patients were analyzed to validate the method. Considering that the most frequent Salmonellaserovar isolated from blood in case of bacteremia is S. typhi, the polymerase chain reaction‐microtiter plate hybridization technique could be used as a novel, rapid diagnostic method for typhoid fever, particularly when standard culture assays are negative.

PCR-single-strand conformation polymorphism (PCR-SSCP) analysis is a convenient technique for the detection of mutations. As the mobility of single-stranded DNA is sequence-dependent it could therefore be used to determine serotype-related sequence variations inListeria monocytogenes. Sero-specific patterns were observed in differentL. monocytogenesserogroups.

Primers for the iap gene were used to develop and optimise a PCR technique for detecting Listeria monocytogenes in meat. The use of these primers gave good results in the detection of L monocytogenes in either artificially or naturally contaminated foodstuffs. The lowest sensitivity limit achieved with PCR was 10¹–10² cells ml^-1 or g^-1 product, either in pure cultures or in artificially and naturally contaminated meat. The PCR method devised has high sensitivity and specificity. It appears to give good results and is very easy and fast to perform, requiring only 30 h to detect Listeria from meat, whereas, conventional methods require almost 96–120 h. © 1997 SCI.

The development of a rapid method for the identification of Listeriaspp. is described. It is based on the polymerase chain reaction amplification of a small fragment from the 16S rRNA gene followed by temperature gradient gel electrophoresis. Forty-five strains of Listeriaspp. (Listeria monocytogenes, Listeria innocua, Listeria ivanovii, Listeria seeligeri, and Listeria welshimeri) were used for the optimization of the protocol. No differences were observed between the results of the identification of the strains tested using traditional methods and those obtained by polymerase chain reaction–temperature gradient gel electrophoresis analysis.

In order to improve the diagnosis ofListeria monocytogenesinfection, we have developed a polymerase chain reaction (PCR)-based assay combined with microplate capture hybridization technique. The system is based on selective amplification ofL. monocytogeneswith two specific primers based on theiapgene. The amplicon produced, with digoxigenin 11-dUTP incorporated during PCR, is hybridized in streptavidin-coated microtitre plates prepared with biotinylated specific DNA probe. The method involved requires approximately 6–8 h, and its high sensitivity, rapidity and simplicity should make it valuable for diagnosis and for epidemiological studies of listeriosis.

A polymerase chain reaction based test was developed for the detection of Salmonella spp. in blood specimens. After amplification of a 389 bp-polymerase chain reaction product from the invA gene, a microtiter plate hybridization assay was performed. The protocol described allowed the detection of six to seven copies of the Salmonella typhi genome, as determined by serial dilutions of DNA from S. typhi. Eighteen blood specimens from artificially infected rats and 22 blood specimens from patients were analyzed to validate the method. Considering that the most frequent Salmonella serovar isolated from blood in case of bacteremia is S. typhi, the polymerase chain reaction-microtiter plate hybridization technique could be used as a novel, rapid diagnostic method for typhoid fever, particularly when standard culture assays are negative.

A combined polymerase chain reaction and restriction endonuclease (RE) enzyme assay was developed to discriminate between Campylobacter coli and Campylobacter jejuni. Amplimers of the FlaA gene obtained by PCR were digested with AluI and HinfI to distinguish C. coli from C. jejuni. With AluI digestion C. jejuni-specific bands were observed at 110, 140 and 160 bp and C. coli-specific bands at 293 and 147 bp. C. jejuni-specific bands of 349 and 109 bp were found by HinfI digestion but HinfI did not digest the FlaA amplimer of C. coli. This combined technique is fast and easy to perform, and distinguishes the two campylobacters unequivocally.