Activity of natural occurring entomopathogenic fungi on nymphal and adult stages of Philaenus spumarius.

The spittlebug Philaenus spumarius (Hemiptera: Aphrophoridae) is the predominant vector of Xylella fastidiosa (Xanthomonadales: Xanthomonadaceae) in Apulia, Italy and the rest of Europe. Current control strategies of the insect vector rely on mechanical management of nymphal stages and insecticide application against adult populations. Entomopathogenic fungi (EPF) are biological control agents naturally attacking spittlebugs and may effectively reduce population levels of host species. Different experimental trials in controlled conditions have been performed to i) identify naturally occurring EPF on P, spumarius in Northwestern Italy, and ii) evaluate the potential for biocontrol of the isolated strains on both nymphal and adult stages of the spittlebug. Four EPF species were isolated from dead P. spumarius collected in semi-field conditions: Beauveria bassiana, Conidiobolus coronatus, Fusarium equiseti and Lecanicillium aphanocladii. All the fungal isolates showed entomopathogenic potential against nymphal stages of P. spumarius (≈ 45 % mortality), except for F. equiseti, in preliminary trials. No induced mortality was observed on adult stage. Lecanicillium aphanocladii was the most promising fungus and its pathogenicity against spittlebug nymphs was further tested in different formulations (conidia vs blastospores) and with natural adjuvants. Blastospore formulation was the most effective in killing nymphal instars and reducing the emergence rate of P, spumarius adults, reaching mortality levels (90%) similar to those of the commercial product Naturalis®, while no or adverse effect of natural adjuvants was recorded. The encouraging results of this study pave way for testing EPF isolates against P, spumarius in field conditions and find new environmentally friendly control strategies against insect vectors of X. fastidiosa.

Technological, functional and safety properties of lactobacilli isolates from soft wheat sourdough and their potential use as antimould cultures.

Bakery products are a common medium for fungal growth due to their high-water activity and nutrients availability. The application of lactic acid bacteria (LAB) isolated from wheat bran or other cereals has shown great potential in controlling the growth of spoilage fungi, guarantee quality and prolong the shelf life of bakery products. This study outlines the antifungal, technological, functional and safety properties of autochthonous LAB microbiota isolated from type 0 soft wheat sourdough fermentation. Antifungal activity of 77 LAB belonging to Lactiplantibacillus plantarum and Lacticaseibacillus casei species isolated from spontaneous sourdough fermentation was tested in vitro against 16 spoilage fungi. Our findings demonstrated that the antifungal activity, enzymatic and safety properties of LAB isolates vary strain-dependently. Four LAB isolates (Lp. plantarum A16, A25, B11, and B15) showed the best traits, in particular strong antifungal activity and good capabilities to produce exopolysaccharides from different carbon sources in vitro. Care should be taken when using Lp. plantarum A310 and B18 and Lc. casei A23, as starter cultures, since these isolates exhibited a multiple antibiotic-resistance. Here we showed the promising potential of different LAB isolates as bio-preservative agents and to provide new insights regarding their prospective use as starter cultures to guarantee safety and palatability.

Antifungal activity of yeasts and lactic acid bacteria isolated from cocoa bean fermentations.

The aim of this study was to select yeast and LAB strains to be used as a mean of biological control against fungal growth during cocoa fermentation process. Their antifungal activity was assessed against six spoilage fungi isolated from both fermented and dried cocoa beans and belonging to Aspergillus and Penicillium genera. An initial screening was carried out by using the overlay method where the plates were examined for fungi inhibition zone around LAB and yeast streaks. Then, the most active strains were studied in inhibition test in 96-well microplates where mould growth was measured by microplate reader at 490 nm. The nature of their antifungal strenght (organic acid and/or proteins) was also evaluated. The most promising candidates as biological agents belonged to the species Lactobacillus fermentum, Lactobacillus plantarum, Saccharomyces cerevisiae and Candida ethanolica and their antifungal strength was attributed mainly to organic acid production (for LAB) and proteinaceous compounds (for yeasts) or to their synergic effect.

Dynamics and Biodiversity of Bacterial and Yeast Communities during Fermentation of Cocoa Beans.

Forastero hybrid cocoa bean fermentations have been carried out in a box (B) and in a heap (H), with or without the inoculation of Saccharomyces cerevisiae and Torulaspora delbrueckii as starter cultures. The bacteria, yeasts, and microbial metabolites (volatile and nonvolatile organic compounds) were monitored during fermentation to assess the connection between microbiota and the release of metabolites during this process. The presence of starter cultures was detected, by means of culture-dependent analysis, during the first 2 days of both fermentations. However, no statistical difference was observed in any of the physicochemical or microbiological analyses. Plate counts revealed the dominance of yeasts at the beginning of both fermentations, and these were followed by acetic acid bacteria (AAB) and lactic acid bacteria (LAB). Hanseniaspora opuntiae, S. cerevisiae, Pichia pijperi, Acetobacter pasteurianus, and Lactobacillus fermentum were the most abundant operational taxonomic units (OTUs) during both fermentation processes (B and H), although different relative abundances were observed. Only the diversity of the fungal species indicated a higher level of complexity in the B fermentations than in the H fermentations (P < 0.05), as well as a statistically significant difference between the initially inoculated starter cultures (P < 0.01). However, the microbial metabolite analysis indicated different distributions of the volatile and nonvolatile compounds between the two procedures, that is, B and H (P < 0.05), rather than between the inoculated and noninoculated fermentations. The box fermentations showed faster carbohydrate metabolism and greater production of organic acid compounds, which boosted the formation of alcohols and esters, than did the heap fermentations. Overall, the microbial dynamics and associations between the bacteria, yeasts, and metabolites were found to depend on the type of fermentation.IMPORTANCE In spite of the limited effectiveness of the considered inoculated starter strains, this study provides new information on the microbial development of box and heap cocoa fermentations, under inoculated and noninoculated conditions, as we coupled yeast/bacterial amplicon-based sequencing data with microbial metabolite detection. The information so far available suggests that microbial communities have played an important role in the evolution of aroma compounds. Understanding the pathways that microorganisms follow during the formation of aromas could be used to improve the fermentation processes and to enhance chocolate quality.

Sausage fermentation and starter cultures in the era of molecular biology methods.

Fermented sausages have a long tradition originating from Europe and they constitute a significant part of the Mediterranean diet. This kind of products has a specific microbiota that is typical of the region or area where they are produced. Therefore, in order to protect the traditional aspect of these products, it is essential to understand the microbial ecology during fermentation by studying the dynamic changes that occur and to select autochthonous starter cultures that can be used in the production. In this paper we summarize the state of the art concerning the selection and use of starter cultures and ecology aspects of naturally fermented sausages. We pay particular attention to the application of bacteriocinogenic strains as they could provide an additional tool in the prevention of foodborne pathogens as well as enhancing the competitiveness of the starter organisms. Microbial ecology of fermented sausages has been determined by traditional microbiological methods, but the introduction in food microbiology of new molecular techniques complements the studies carried out so far and allows scientists to overcome the limitations of traditional methods. Next Generation Sequencing (NGS) techniques represent a change in the way microbiologists address ecology and diversity in foods. Indeed the application of metataxonomics and metagenomics will permit a detailed understanding of microbial ecology. A thorough knowledge of the mechanisms behind the biological processes will enhance meat fermentation control and modulation to obtain products with desired organoleptic properties.

Study of Lactococcus lactis during advanced ripening stages of model cheeses characterized by GC-MS.

Lactococcus lactis, is extensively used as starter culture in dairy products. Nevertheless, it has recently been detected in cheese, as metabolically active cells, in advanced ripening stages. In this study, we assessed the viability of L. lactis subsp. lactis in model cheeses up to 180 days of ripening by both culture-dependent and -independent methods. In addition, we studied the expression of metC and als genes involved in the production of aroma compounds detected by Gas Chromatography-Mass Spectrometry (GC-MS). Three L. lactis subsp. lactis commercial starters were inoculated in pasteurized milk and model cheeses were manufactured and ripened for six months. Samples were analysed at manufacturing and ripening steps, in terms of viability of L. lactis by both traditional plating and direct analysis of RNA by reverse transcription quantitative PCR (RT-qPCR), and in terms of aroma profile by GC-MS. Relatively to RT-qPCR analysis, L. lactis was found viable throughout the whole process of cheesemaking and aging, with final average loads of 3-4 Log CFU/g at 180 days. On the contrary, the microorganism was not detected, in ripened samples, by traditional plating on M17 medium, suggesting its entering in a viable but not cultivable (VBNC) state. The aroma profiles of the cheeses highlighted the presence of volatile compounds related to cheese flavor as acetoin, diacetyl, 2,3-butanediol and dimethyl disulfide, whose presence was partially correlated to metC and als genes expression. These results add new insights on the capability of L. lactis to persist during late cheese ripening and suggest a potential contribution of the microorganism to cheese flavor formation.

Direct Application of Rep-PCR on Type I Sourdough Matrix to Monitor the Dominance and Persistence of a Lactobacillus plantarum Starter Throughout Back-Slopping.

This study describes the optimization and application of repetitive element-PCR (rep-PCR) technique directly on microbial DNA extracted from type I sourdoughs for fast monitoring of a Lb. plantarum starter strain (P1FMC) throughout daily back-slopping. The challenge was to follow and study the performance of a starter culture directly in sourdoughs without cultivation on selective media. The extraction of good quality microbial DNA suitable for amplification from a complex matrix such as dough was the first target. In addition, the objective to obtain a clear rep-PCR profile referable to a specific starter strain among a microbial community was pursued. Co-inoculum trials, in flour matrix, with Lb. plantarum P1FMC and L. lactis LC71 strains and, subsequently, type I sourdough back-slopping trials were performed. The rep-PCR amplification profiles obtained were clearly referable to that of Lb. plantarum P1FMC starter in both co-inoculum trials (also when it was present with one order of magnitude less with respect to L. lactis LC71) and back-slopping trials where it dominated the fermentation process with loads of 108 cfu g-1 and prevailed on the autochthonous microbiota. Thus, the approach proposed in this paper could be considered a methodological advancement, based on a culture-independent one-step rep-PCR, suitable for fast monitoring of starter performance.

Impact of Saccharomyces cerevisiae and Torulaspora delbrueckii starter cultures on cocoa beans fermentation.

Aim of this work was to study the impact of mixed cultures of Saccharomyces cerevisiae and Torulaspora delbrueckii and T. delbrueckii monoculture on the fermentation process conducted on two different cocoa hybrids, PS1319 and SJ02, in Bahia, Brazil. This was performed throughout studying physico-chemical changes during the fermentation process and analyzing volatile compounds and sensory analysis of chocolates. (GTG)5-PCR fingerprinting was used to type isolates at strain level allowing to assess the implantation of the starter cultures added. Resulted clusters were composed by T. delbrueckii strains isolated during the first 24h of fermentation. On the contrary, S. cerevisiae, the most strongly fermenting ethanol-tolerant species, took over the fermentation at a second stage. Quantification data of T. delbrueckii during spontaneous fermentation confirm the attitude of this species of not being so commonly involved in this process. This study also showed that the inoculum influenced the PS1319 hybrid end-product quality, changing analytic profile and sensory perception of chocolates. No big influences were recorded for SJ02 hybrid, but this may be improved. In combination with S. cerevisiae, T. delbrueckii had a positive influence on the analytical profile of chocolates. The application of starter cultures did change the aroma profile of the resulting chocolate as determined by GC-MS; in some case the differences observed had a significantly impact on the consumer perception of the chocolates.

Fate of Lactococcus lactis starter cultures during late ripening in cheese models.

The presence of Lactococcus lactis, commonly employed as starter culture, was, recently, highlighted and investigated during late cheese ripening. Thus, the main goal of the present study was to assess the persistence and viability of this microorganism throughout manufacturing and ripening of model cheeses. Eight commercial starters, constituted of L. lactis subsp. lactis and L. lactis subsp. cremoris, were inoculated in pasteurized milk in order to manufacture miniature cheeses, ripened for six months. Samples were analysed at different steps (milk after inoculum, curd after cutting, curd after pressing and draining, cheese immediately after salting and cheese at 7, 15, 30, 60, 90, 120, 150 and 180 days of ripening) and submitted to both culture-dependent (traditional plating on M17) and -independent analysis (reverse transcription-quantitative PCR). On the basis of direct RNA analysis, L. lactis populations were detected in all miniature cheeses up to the sixth month of ripening, confirming the presence of viable cells during the whole ripening process, including late stages. Noteworthy, L. lactis was detected by RT-qPCR in cheese samples also when traditional plating failed to indicate its presence. This discrepancy could be explain with the fact that lactococci, during ripening process, enter in a stressed physiological state (viable not culturable, VNC), which might cause their inability to grow on synthetic medium despite their viability in cheese matrix. Preliminary results obtained by "resuscitation" assays corroborated this hypothesis and 2.5% glucose enrichment was effective to recover L. lactis cells in VNC state. The capability of L. lactis to persist in late ripening, and the presence of VNC cells which are known to shift their catabolism to peptides and amino acids consumption, suggests a possible technological role of this microorganism in cheese ripening with a possible impact on flavour formation.

Molecular identification and physiological characterization of yeasts, lactic acid bacteria and acetic acid bacteria isolated from heap and box cocoa bean fermentations in West Africa.

Yeast, lactic acid bacteria (LAB) and acetic acid bacteria (AAB) populations, isolated from cocoa bean heap and box fermentations in West Africa, have been investigated. The fermentation dynamicswere determined by viable counts, and 106 yeasts, 105 LAB and 82 AAB isolateswere identified by means of rep-PCR grouping and sequencing of the rRNA genes. During the box fermentations, the most abundant species were Saccharomyces cerevisiae, Candida ethanolica, Lactobacillus fermentum, Lactobacillus plantarum, Acetobacter pasteurianus and Acetobacter syzygii, while S. cerevisiae, Schizosaccharomyces pombe, Hanseniaspora guilliermondii, Pichia manshurica, C. ethanolica, Hanseniaspora uvarum, Lb. fermentum, Lb. plantarum, A. pasteurianus and Acetobacter lovaniensis were identified in the heap fermentations. Furthermore, the most abundant species were molecularly characterized by analyzing the rep-PCR profiles. Strains grouped according to the type of fermentations and their progression during the transformation process were also highlighted. The yeast, LAB and AAB isolates were physiologically characterized to determine their ability to grow at different temperatures, as well as at different pH, and ethanol concentrations, tolerance to osmotic stress, and lactic acid and acetic acid inhibition. Temperatures of 45 °C, a pH of 2.5 to 3.5, 12% (v/v) ethanol and high concentrations of lactic and acetic acid have a significant influence on the growth of yeasts, LAB and AAB. Finally, the yeastswere screened for enzymatic activity, and the S. cerevisiae, H. guilliermondii, H. uvarumand C. ethanolica species were shown to possess several enzymes that may impact the quality of the final product.

Detection and viability of Lactococcus lactis throughout cheese ripening.

Recent evidences highlighted the presence of Lactococcus lactis during late cheese ripening. For this reason, the role of this microorganism, well known as dairy starter, should be reconsidered throughout cheese manufacturing and ripening. Thus, the main objective of this study was to develop a RT-qPCR protocol for the detection, quantification and determination of the viability of L. lactis in ripened cheese samples by direct analysis of microbial nucleic acids. Standard curves were constructed for the specific quantification of L. lactis in cheese matrices and good results in terms of selectivity, correlation coefficient and efficiency were obtained. Thirty-three ripened cheeses were analyzed and, on the basis of RNA analysis, twelve samples showed 106 to 108 CFU of L. lactis per gram of product, thirteen from 103 to 105 CFU/g, and in eight cheeses, L. lactis was not detected. Traditional plating on M17 medium led to loads ranging from 105 to 109 CFU/g, including the cheese samples where no L. lactis was found by RT-qPCR. From these cheeses, none of the colonies isolated on M17 medium was identified as L. lactis species. These data could be interpreted as a lack of selectivity of M17 medium where colony growth is not always related to lactococcal species. At the same time, the absence or low abundance of L. lactis isolates on M17 medium from cheese where L. lactis was detected by RT-qPCR support the hypothesis that L. lactis starter populations are mainly present in viable but not culturable state during ripening and, for this reason, culture-dependent methods have to be supplemented with direct analysis of cheese.

Endogenous isoflavone methylation correlates with the in vitro rooting phases of Spartium junceum L. (Leguminosae).

Spartium junceum L. (Leguminosae) is a perennial shrub, native to the Mediterranean region in southern Europe, widespread in all the Italian regions and, as a leguminous species, it has a high isoflavone content. An in vitro culture protocol was developed for this species starting from stem nodal sections of in vivo plants, and isoflavone components of the in vitro cultured tissues were studied by means of High Performance Liquid Chromatography (HPLC) analytical techniques. Two main isoflavones were detected in the S. junceum tissues during the in vitro propagation phases: Genistein (4',5,7-Trihydroxyisoflavone), already reported in this species, and its methylated form 4',5,7-Trimethoxyisoflavone, detected for the first time in this plant species (0.750 ± 0.02 mg g(-1) dry tissue). The presence of both of these compounds in S. junceum tissues was consistently detected during the in vitro multiplication phase. The absence of the methylated form within plant tissues in the early phases of the in vitro adventitious root formation was correlated with its negative effect displayed on root induction and initiation phases, while its presence in the final "root manifestation" phase influenced positively the rooting process. The unmethylated form, although detectable in tissues in the precocious rooting phases, was no longer present in the final rooting phase. Its effect on rooting, however, proved always to be beneficial.

rRNA-based monitoring of the microbiota involved in Fontina PDO cheese production in relation to different stages of cow lactation.

Fontina Protected Denomination of Origin (PDO) cheese is a full-fat semi-cooked cheese traditionally made in Northwest Italy (Aosta Valley) and manufactured from raw cow's milk. The management of cattle farms in Aosta Valley calls for seasonal migration to high pastures during the summer and the concentration of calving during the autumn and the beginning of the winter. Based on cattle physiology and given to calving seasonality, three cow lactation phases i.e. post-partum, oestrus and early gestation, can be identified and an effect could be hypothesized on average milk composition and on cheese quality. The aim of the present paper was to investigate the bacterial dynamics during Fontina PDO cheese manufacturing and ripening, in relation to the different lactation stages, in order to evaluate a possible correlation between microbiota and phase of lactation. For this purpose, microbial RNA analysis was carried out by RT-PCR coupled with DGGE and high-throughput sequencing. A good performance of the starter cultures was highlighted throughout Fontina PDO manufacturing and ripening; in fact, the starter prevailed against the autochthonous microbiota. Thus, the microbial activity, which was supposed to affect the final quality of Fontina PDO cheese, appeared to be strictly associated to the presence of the starter, which did not show any difference in its performance according to the different stages of cow lactation. Therefore, the results of this research highlighted a negligible correlation between the microbiota of raw milk and the organoleptic quality and typicity of Fontina cheese in relation to lactation seasonality.

Aerobic deterioration stimulates outgrowth of spore-forming Paenibacillus in corn silage stored under oxygen-barrier or polyethylene films.

The occurrence of Bacillus and Paenibacillus spores in silage is of great concern to dairy producers because their spores can survive pasteurization and some strains are capable of subsequently germinating and growing under refrigerated conditions in pasteurized milk. The objectives of this study were to verify the role of aerobic deterioration of corn silage on the proliferation of Paenibacillus spores and to evaluate the efficacy of oxygen-barrier films used to cover silage during fermentation and storage to mitigate these undesirable bacterial outbreaks. The trial was carried out on whole-crop maize (Zea mays L.) inoculated with a mixture of Lactobacillus buchneri, Lactobacillus plantarum, and Enterococcus faecium. A standard polyethylene film and a polyethylene-polyamide film with an enhanced oxygen barrier were used to produce the silage bags for this experiment. The silos were stored indoors at ambient temperature (18 to 22°C) and opened after 110 d. The silage was sampled after 0, 2, 5, 7, 9, and 14 d of aerobic exposure to quantify the growth of endospore-forming bacteria during the exposure of silages to air. Paenibacillus macerans (gram-positive, facultatively anaerobic bacteria) was able to develop during the aerobic exposure of corn silage. This species was present in the herbage at harvesting, together with clostridial spores, and survived ensiling fermentation; it constituted more than 60% of the anaerobic spore formers at silage opening. During silage spoilage, the spore concentration of P. macerans increased to values greater than 7.0 log10 cfu/g of silage. The use of different plastic films to seal silages affected the growth of P. macerans and the number of spores during aerobic exposure of silages. These results indicate that the number of Paenibacillus spores could greatly increase in silage after exposure to air, and that oxygen-barrier films could help to reduce the potential for silage contamination of this important group of milk spoilage microorganisms by delaying the onset of aerobic deterioration.

Culture independent methods to assess the diversity and dynamics of microbiota during food fermentation.

Culture independent methods first appeared in the food microbiology field at the end of the 90s and since then they have been applied extensively. These methods do not rely on cultivation and target nucleic acids (DNA and RNA) to identify and follow the changes that occur in the main populations present in a specific ecosystem. The method that has most often been used as a culture independent method in food microbiology is denaturing gradient gel electrophoresis (DGGE). The number of papers dealing with DGGE grew exponentially in the late nineties and, by analysing the studies available in the literature, it is possible to describe a trend in the subjects that have been investigated. DGGE was first used as a tool to monitor the ecology of fermented food, such as fermented sausage, cheese and sourdough, and later it also showed its potential in microbial spoilage process. In the last few years, the main application of DGGE has been to study fermented food from Asia, Africa and South America. The information collected using DGGE has made it possible to confirm the existing knowledge on food fermentation and spoilage. However, in some cases, new evidence that helps scientists to fully comprehend a specific microbial ecosystem has emerged. In this review, the roadmap of culture independent methods in food microbiology will be summarized, focusing on the DGGE technique. Examples of how this approach is useful to obtain a better understanding of microbial diversity are reported for several kinds of fermented food, such as fermented sausage, cheese and wine. The future of culture independent methods in food microbiology, with the increasing availability of next generation sequencing techniques, is also discussed.

Cheese surface microbiota complexity: RT-PCR-DGGE, a tool for a detailed picture?

In this work, a culture-independent approach, based on PCR-DGGE and RT-PCR-DGGE, has been used to study the succession of bacterial communities that are encountered in Fontina PDO cheese. As already found for other smear ripened cheeses, it appeared that coryneform bacteria were actively present and could therefore be considered determinant in rind formation. DGGE profiles, especially at the RNA level, have shown the presence of Brevibacterium, Corynebacterium and Arthrobacter genera. RT-PCR-DGGE gels have lead to a richer band profile than the one obtained on the basis of DNA analysis, thus indicating that RNA analysis can highlight bacterial species that DNA analysis is not able to show. Thus, the biodiversity of the Fontina PDO surface has been described better by means of RT-PCR-DGGE, and RNA molecules should be considered a more informative target than DNA.

Candida zemplinina can reduce acetic acid produced by Saccharomyces cerevisiae in sweet wine fermentations.

In this study we investigated the possibility of using Candida zemplinina, as a partner of Saccharomyces cerevisiae, in mixed fermentations of must with a high sugar content, in order to reduce its acetic acid production. Thirty-five C. zemplinina strains, which were isolated from different geographic regions, were molecularly characterized, and their fermentation performances were determined. Five genetically different strains were selected for mixed fermentations with S. cerevisiae. Two types of inoculation were carried out: coinoculation and sequential inoculation. A balance between the two species was generally observed for the first 6 days, after which the levels of C. zemplinina started to decrease. Relevant differences were observed concerning the consumption of sugars, the ethanol and glycerol content, and acetic acid production, depending on which strain was used and which type of inoculation was performed. Sequential inoculation led to the reduction of about half of the acetic acid content compared to the pure S. cerevisiae fermentation, but the ethanol and glycerol amounts were also low. A coinoculation with selected combinations of S. cerevisiae and C. zemplinina resulted in a decrease of ~0.3 g of acetic acid/liter, while maintaining high ethanol and glycerol levels. This study demonstrates that mixed S. cerevisiae and C. zemplinina fermentation could be applied in sweet wine fermentation to reduce the production of acetic acid, connected to the S. cerevisiae osmotic stress response.

Microbial dynamics during aerobic exposure of corn silage stored under oxygen barrier or polyethylene films.

The aims of this study were to compare the effects of sealing forage corn with a new oxygen barrier film with those obtained by using a conventional polyethylene film. This comparison was made during both ensilage and subsequent exposure of silage to air and included chemical, microbiological, and molecular (DNA and RNA) assessments. The forage was inoculated with a mixture of Lactobacillus buchneri, Lactobacillus plantarum, and Enterococcus faecium and ensiled in polyethylene (PE) and oxygen barrier (OB) plastic bags. The oxygen permeability of the PE and OB films was 1,480 and 70 cm³ m⁻² per 24 h at 23°C, respectively. The silages were sampled after 110 days of ensilage and after 2, 5, 7, 9, and 14 days of air exposure and analyzed for fermentation characteristics, conventional microbial enumeration, and bacterial and fungal community fingerprinting via PCR-denaturing gradient gel electrophoresis (DGGE) and reverse transcription (RT)-PCR-DGGE. The yeast counts in the PE and OB silages were 3.12 and 1.17 log10 CFU g⁻¹, respectively, with corresponding aerobic stabilities of 65 and 152 h. Acetobacter pasteurianus was present at both the DNA and RNA levels in the PE silage samples after 2 days of air exposure, whereas it was found only after 7 days in the OB silages. RT-PCR-DGGE revealed the activity of Aspergillus fumigatus in the PE samples from the day 7 of air exposure, whereas it appeared only after 14 days in the OB silages. It has been shown that the use of an oxygen barrier film can ensure a longer shelf life of silage after aerobic exposure.

Biodiversity and dynamics of meat fermentations: the contribution of molecular methods for a better comprehension of a complex ecosystem.

The ecology of fermented sausages is complex and includes different species and strains of bacteria, yeasts and molds. The developments in the field of molecular biology, allowed for new methods to become available, which could be applied to better understand dynamics and diversity of the microorganisms involved in the production of sausages. Methods, such as denaturing gradient gel electrophoresis (DGGE), employed as a culture-independent approach, allow to define the microbial dynamics during the fermentation and ripening. Such approach has highlighted that two main species of lactic acid bacteria, namely Lactobacillus sakei and Lb. curvatus, are involved in the transformation process and that they are accompanied by Staphylococcus xylosus, as representative of the coagulase-negative cocci. These findings were repeatedly confirmed in different regions of the world, mainly in the Mediterranean countries where dry fermented sausages have a long tradition and history. The application of molecular methods for the identification and characterization of isolated strains from fermentations highlighted a high degree of diversity within the species mentioned above, underlining the need to better follow strain dynamics during the transformation process. While there is an important number of papers dealing with bacterial ecology by using molecular methods, studies on mycobiota of fermented sausages are just a few. This review reports on how the application of molecular methods made possible a better comprehension of the sausage fermentations, opening up new fields of research that in the near future will allow to unravel the connection between sensory properties and co-presence of multiple strains of the same species.

Evolution of chemico-physical characteristics during manufacture and ripening of Castelmagno PDO cheese in wintertime.

Biochemical, volatile and textural profiles during manufacture and ripening were determined in samples of Castelmagno PDO cheese obtained from three different batches in the main artisan cheese plant of Castelmagno PDO production area. At the end of manufacture, samples were characterised by a pH of 6.57% and 52.4% moisture content. The HPLC analysis of organic acids and sugars showed the exhaustion of lactose content, while Urea-PAGE indicated extensive primary proteolysis of both ß-casein and αs1-casein. During ripening, cheeses were characterised by high degradation of ß-casein and αs1-casein, due to bacterial action. RP-HPLC profiles showed a high production of peptides eluted between 20 and 30min. In total, 92 volatile compounds were identified in cheese headspace. Texture profiles showed an increase in hardness, gumminess, chewiness and adhesiveness values, as well as a decrease in cohesiveness during ripening.