A promising eco-sustainable wound dressing based on cellulose extracted from Spartium junceum L. and impregnated with Glycyrrhiza glabra L extract: Design, production and biological properties.

Glycyrrhiza glabra extract is widely known for its antioxidant and anti-inflammatory properties and can improve the wound healing process. The aim of this work was to shorten the time of the healing process by using an eco-sustainable wound dressing based on Spanish broom flexible cellulosic fabric by impregnation with G. glabra extract-loaded ethosomes. Chemical analysis of G. glabra extract was performed by LC-DAD-MS/MS and its encapsulation into ethosomes was obtained using the ethanol injection method. Lipid vesicles were characterized in terms of size, polydispersity index, entrapment efficiency, zeta potential, and stability. In vitro release studies, biocompatibility, and scratch test on 3T3 fibroblasts were performed. Moreover, the structure of Spanish broom dressing and its ability to absorb wound exudate was characterized by Synchrotron X-ray phase contrast microtomography (SR-PCmicroCT). Ethosomes showed a good entrapment efficiency, nanometric size, good stability over time and a slow release of polyphenols compared to the free extract, and were not cytotoxic. Lastly, the results revealed that Spanish broom wound dressing loaded with G. glabra ethosomes is able to accelerate wound closure by reducing wound healing time. To sum up, Spanish broom wound dressing could be a potential new green tool for biomedical applications.

Spray-dried chestnut extract containing Lactobacillus rhamnosus cells as novel ingredient for a probiotic chestnut mousse.

AIMS: Consumers' demand for innovative probiotic products has recently increased. In previous studies, chestnuts were evaluated as substrate for the growth of lactobacilli and chestnut extract was found to enhance acid tolerance of probiotic strains. The main objectives of this study were to evaluate the suitability of chestnut extract as carrier for spray drying of two probiotic Lactobacillus rhamnosus strains and to develop a probiotic food chestnut based. METHODS AND RESULTS: The optimal settings for the spray-drying processes were defined and the loads of undamaged cells in the dried powders were quantified. Spray-dried cultures were incorporated into an anhydrous basis for chestnut mousse developed ad hoc. In this form, viable cells remained stable over 10(8) CFU g(-1) during a 3 months long storage at 15°C. Sensorial analysis did not highlighted significant differences (P < 0·05) in preference between probiotic-supplemented and control mousses. CONCLUSIONS: Results suggest that chestnut mousse, a food product naturally rich in antioxidant compounds, may represent an excellent carrier for probiotics delivering. SIGNIFICANCE AND IMPACT OF THE STUDY: To authors' knowledge, this is the first information on the survival of lactobacilli in an anhydrous basis for dessert.

Production of fermented chestnut purees by lactic acid bacteria.

The objective of this study was to develop a new chestnut-based puree, in order to seasonally adjust the offer and use the surplus of undersized production, providing, at the same time, a response to the growing demand for healthy and environmentally friendly products. Broken dried chestnuts have been employed to prepare purees to be fermented with six different strains of Lactobacillus (Lb.) rhamnosus and Lactobacillus casei. The fermented purees were characterized by a technological and sensorial point of view, while the employed strains were tested for their probiotic potential. Conventional in vitro tests have indicated the six lactobacilli strains as promising probiotic candidates; moreover, being the strains able to grow and to survive in chestnut puree at a population level higher than 8 log10 CFU/mL along 40 days of storage at 4 °C, the bases for the production of a new food, lactose-free and with reduced fat content, have been laid.

Short communication: technological and genotypic comparison between Streptococcus macedonicus and Streptococcus thermophilus strains coming from the same dairy environment.

The species Streptococcus thermophilus is widely used for the preparation of several dairy products, and its technological contribution is clear. On the other hand, although Streptococcus macedonicus was first described more than 10 yr ago and, despite the scientific interest around this issue, the exact role of Strep. macedonicus in cheese making has yet to be clarified. In this study, 121 strains belonging to both species and isolated from the same dairy environment were genetically characterized by random amplification of polymorphic DNA (RAPD)-PCR and compared for the main biochemical features of technological interest, such as acid production, galactose utilization, citrate metabolism, exopolysaccharide production, and lipolytic, ureolytic, exocellular proteolytic, and decarboxylasic activities. Analysis by RAPD-PCR highlighted a remarkable genotypic heterogeneity among strains in both species, and, at a similarity level of 78%, all the isolates and reference strains of Strep. thermophilus grouped together and were well separated from the strains of Strep. macedonicus, confirming that these 2 species are different microbial entities. Comparison between genetic and phenotypic or biotechnological data did not reveal any relationships.

Could halophilic archaea improve the traditional salted anchovies (Engraulis encrasicholus L.) safety and quality?

AIMS: The positive influence of two selected extremely halophilic archaea strains in the production of salted anchovies (Engraulis encrasicolus, L., 1758) was highlighted. METHODS AND RESULTS: Anchovies produced with salt artificially contaminated with halophiles exhibited lower loads of staphylococci, Enterobacteriaceae and lactic acid bacteria, and a reduced content of histamine as well as an improved organoleptic acceptance. CONCLUSIONS: The findings of this survey are expected to enhance the safety of salted anchovies, with regard to the histamine formation during ripening, and to improve the sensory attributes of this product. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents the first report on the positive influence of halophilic archaea in traditional salted anchovies production, thus suggesting new perspectives about a conscious employment of properly selected haloarchaea strains in this traditional manufacture.

Molecular and physiological characteristics of a grape yeast strain containing atypical genetic material.

The knowledge about wine yeasts remains largely dominated by the extensive studies on Saccharomyces (S.) cerevisiae. Molecular methods, allowing discrimination of both species and strains in winemaking, can profitably be applied for characterization of the microflora occurring in winemaking and for monitoring the fermentation process. Recently, some novel yeast isolates have been described as hybrid between S. cerevisiae and Saccharomyces species, leaving the Saccharomyces strains containing non-Saccharomyces hybrids essentially unexplored. In this study, we have analyzed a yeast strain isolated from "Primitivo" grape (http://www.ispa.cnr.it/index.php?page=collezioni&lang=en accession number 12998) and we found that, in addition to the S. cerevisiae genome, it has acquired genetic material from a non-Saccharomyces species. The study was focused on the analysis of chromosomal and mitochondrial gene sequences (ITS and 26S rRNA, SSU and COXII, ACTIN-1 and TEF), 2D-PAGE mitochondrial proteins, and spore viability. The results allowed us to formulate the hypothesis that in the MSH199 isolate a DNA containing an rDNA sequence from Hanseniaspora vineae, a non-Saccharomyces yeast, was incorporated through homologous recombination in the grape environment where yeast species are propagated. Moreover, physiological characterization showed that the MSH199 isolate possesses high technological quality traits (fermentation performance) and glycerol production, resistance to ethanol, SO2 and temperature) useful for industrial application.

Short communication: Identification and technological characterization of yeast strains isolated from samples of water buffalo Mozzarella cheese.

Sixty yeast cultures were isolated from samples of water buffalo Mozzarella, a popular "pasta filata" cheese, originating on 16 farms located in the provinces of Salerno, Caserta, and Frosinone (Italy). Strains were identified by means of 5.8S internal transcribed spacer rDNA PCR-RFLP combined with 26S rRNA gene partial sequencing and characterized for their ability to exert biochemical properties of technological interest. The recorded dominance of fermenting yeasts such as the lactose-fermenting Kluyveromyces marxianus (38.3% of the total isolates) and the galactose-fermenting Saccharomyces cerevisiae (21.6% of the total isolates) suggests that these yeasts contribute to the organoleptic definition of the water buffalo Mozzarella. The speciographic analysis revealed the presence of 7 other species rarely or never reported in a dairy environment belonging to the genera Pichia and Candida, whose role in Mozzarella cheese organoleptic properties need to be further investigated.

Heterotrophic microorganisms in deteriorated medieval wall paintings in southern Italian churches.

The Campania region in southern Italy is noted for its large number of churches that harbour invaluable frescoes, dated from the beginnings of the 4th up to the 13th century. The wall paintings represent an integral part of the monuments, and their deterioration constitutes a potentially significant loss for the world's cultural heritage. Heterotrophic microorganisms such as bacteria and mould can grow on the surface of paintings that contain a wide range of organic and inorganic constituents, and provide different ecological niches that are exploited by a large variety of microbial species. We isolated and identified the heterotrophic microorganisms found in the biodegraded medieval wall paintings of seven historical churches in Campania. The paintings showed different levels of microbial contamination. Microbiological analysis of different paintings gave an overview of the different heterotrophic microorganisms. Bacteria and moulds were isolated from 77% of the sampling points analysed, in which the most common type of alteration was discolouration often associated with detachment of the paint layer. Bacterial strains were identified by 16S rRNA partial sequence analysis. The Bacillus genus was isolated in all churches, even though the type of species was variable, whereas all actinomycetes strains, isolated in five of the seven churches analysed, could be referred to the Streptomyces genus. The similarity of the sequences analysed of the 42 Bacillus spp., 2 Paenibacillus spp. and reference strains of different species showed that these bacteria differentiated in 14 groups. The most frequently occurring taxa were most closely related to Bacillus cereus/thurigiensis/anthracis and Bacillus pumilus groups. Thirteen Streptomyces spp. were differentiated in seven groups on the basis of neighbor-joining analysis of 16S rRNA. Fungi belonging to the genera Penicillium, Aspergillus, Fusarium and Alternaria were also isolated from deteriorated wall paintings.

Isolation of Saccharomyces cerevisiae strains from different food matrices and their preliminary selection for a potential use as probiotics.

AIMS: To isolate acid- and bile-resistant Saccharomyces cerevisiae strains directly from food samples and to preliminarily select them on the basis of fundamental probiotic properties. METHODS AND RESULTS: A rapid screening method allowed the isolation and selection of 20 acid- and bile-resistant yeasts from foods, avoiding time-consuming isolation steps. The strains were characterized for their specific survival in simulated gastric juice and in intestinal fluid after pre-exposure at low pH. Ten isolates demonstrated a satisfactory survival percentage in intestinal fluid after pre-exposure to gastric juice and appreciable lipolytic and proteolytic properties, as demonstrated by the API-ZYM test. By using molecular methods five strains were identified as Saccharomyces cerevisiae, three as Candida spp., one as Candida pararugosa and one as Pichia spp. The Saccharomyces cerevisiae strains showed considerable probiotic properties, achieving a 80< % <90 survival through the simulated gastrointestinal tract, as well as interesting glucosidase activities. CONCLUSIONS: The research represents an efficient strategy to select and identify Saccharomyces cerevisiae strains with desirable acid and bile resistances. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper reports the direct selection of potentially probiotic yeasts from foods and provides indications about the ability of Saccharomyces cerevisiae strains to survive conditions simulating the human gastrointestinal tract.

Response of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella typhimurium, and Staphylococcus aureus to the thermal stress occurring in model manufactures of Grana Padano cheese.

The purpose of this research was to investigate the effect of temperature in the technology of production of Grana cheese against Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Staphylococcus aureus. According to the technology of production, the cheese curds are cooked at 55 degrees C and then cooled at room temperature (25 degrees C). A curd-cooling model was developed to estimate the temperature variation across the curd during cooling, and the thermal stress was applied to the pathogens according to the model in model-scale productions of Grana cheese artificially contaminated with approximately 10(4) cfu/mL of the selected pathogens. According to the numerical results, the initial temperature inside the cheese is kept at almost the initial value (above 50 degrees C) for at least 4 h during cooling, whereas the crust of the curd cools rapidly to 30 degrees C in the first hour. The best case was that of the core of the cheese where the high temperature was able to efficiently eliminate the contaminating pathogens. Moreover, the worst case was where the external ring of the curd in which a more rapid cooling allowed bacterial survival. Therefore, the thermal stress in the technology of production of Grana cheese can be only partially effective in the control of the selected pathogens. However, the whole technology of production includes other hurdles that can affect the survival of the pathogens and that need to be taken into account as a whole to evaluate the safety of Grana Padano cheese.

Presence and characterisation of verotoxin producing E. coli in fresh Italian pork sausages, and preparation and use of an antibiotic-resistant strain for challenge studies.

One hundred and twenty six samples of fresh pork sausages were analysed for the presence of verocytotoxigenic Escherichia coli (VTEC). Selective enrichment followed by DNA extraction and PCR amplification of the stx1 and stx2 genes highlighted the occurrence of the above mentioned genes in 20 out of 126 samples screened. From the stx positive enriched cultures, isolation was performed on CT-SMAC agar plates after immuno-magnetic separation of E. coli O157. Fifty three non-sorbitol fermenting isolates were obtained and further characterised, along with the reference strain E. coli ATCC 35150(T). All the isolates were characterised by PCR assays, assessing the presence of stx1, stx2, rfbE(O157:H7), eae and hlyA genes. The overall prevalence of VTEC was found to be 16%. VTEC strains were also characterised by plasmid profiling and REA-PFGE analysis, which allowed strain clustering into 5 and 8 groups, respectively. In addition, an antibiotic resistant E. coli O157:H7 strain was selected and used in challenge tests of raw pork at 4°C. This strain could be selectively counted in the presence of a normal background microflora and it was shown that it could survive for 1week at 4°C in the raw food studied.

Inter-simple sequence repeat markers and flow cytometry for the characterization of closely related Citrus limon germplasms.

The genetic relationship among commercial cultivars of Citrus limon (lemon) was analysed by inter-simple sequence repeats (ISSR) and flow cytometry techniques. Two cultivars with a close germplasm were distinguished by screening 10 SSR primers and by measuring DNA content of prestained nuclei.

PCR detection of staphylococcal enterotoxin genes in Staphylococcus spp. strains isolated from meat and dairy products. Evidence for new variants of seG and seI in S. aureus AB-8802.

AIMS: Evaluation of the occurrence of most known staphylococcal enterotoxin (SE) genes, egc (enterotoxin gene cluster) and TSST1 (toxic shock syndrome toxin 1) gene in both coagulase-positive (CPS) and coagulase-negative (CNS) staphylococcal strains isolated from meat and dairy products. METHODS AND RESULTS: Specificity and reliability of the PCR detection methods used were ascertained by using nine reference strains of Staphylococcus (S. aureus) harbouring SE genes (seA to seE; seG, seH, seI, seM, seJ, seN and seO) and egc (containing the following sequence of genes: seO, seM, seI, phient1, phient2, seN and seG). Of 109 wild Staphylococcus spp. strains analysed, only 11 S. aureus strains were SE and/or TSST1 PCR-positive. The last 11 strains also appeared to harbour the egc. Restriction endonuclease analysis of part of the egc of both reference and wild strains showed that different variants of the egc exist. Moreover, nucleotide sequences of seG and seI indicate that the egc of the strain AB-8802 is characterized by the presence of variants of these enterotoxins (seGv and seIv). CONCLUSIONS: The occurrence of SE genes in CNS and other non-S. aureus species isolated from Napoli-type salami, raw water buffalo milk and natural whey cultures used for mozzarella cheese manufacturing is very rare. SIGNIFICANCE AND IMPACT OF THE STUDY: During this study it was shown that at least five different egc may exist in S. aureus. A thorough study of egc polymorphism should provide further insight into the phylogenetics of the egc.

Diversity and dynamics of communities of coagulase-negative staphylococci in traditional fermented sausages.

AIMS: Evaluation of composition and evolution of the coagulase-negative staphylococci (CNS) communities in two traditionally fermented sausages (salsiccia and soppressata lucana) produced in Basilicata, southern Italy. METHODS AND RESULTS: A culture-dependent approach based on isolation on selective media and identification with phenotypic and molecular methods was used. Phenotypic data of 471 strains were analysed by multivariate statistical methods by using 28 strains from culture collections and 48 strains identified by molecular methods (such as 16S rDNA sequencing, species-specific PCR assays, intergenic spacer region-PCR and PCR-denaturing gradient gel electrophoresis) as a reference. The CNS microflora of the sausages was found to be dominated by different biotypes of Staphylococcus xylosus (51.2%), followed by S. pulvereri/vitulus, S. equorum and S. saprophyticus (13.4, 10.2 and 10%, respectively). Other species (S. succinus, S. pasteuri, S. epidermidis, S. warneri and Macrococcus caseolyticus) were also present at lower levels. Identification of 25% of the isolates was impossible. CONCLUSIONS: The composition of CNS communities varied significantly with sausage type, plant and ripening time and clear differences were found among communities of salsiccia and soppressata at the end of ripening. SIGNIFICANCE AND IMPACT OF THE STUDY: Phenotypic characterization, supported by molecular and statistical analyses, can be considered a useful approach for typing a large number of isolates and for monitoring the evolution of staphylococcal communities during sausage fermentation but does not always provide a satisfactory identification of the isolates.

PCR-based detection of enterotoxigenic Staphylococcus aureus in the early stages of raw milk cheese making.

AIMS: To define PCR-based detectability of Staphylococcus aureus in raw milk and intermediate products of raw milk cheese making in the presence of a complex background microflora by targetting different specific genes harboured by a single strain. METHODS AND RESULTS: The strain Staph. aureus FRI 137 harbouring nuc, sec, seg, seh and sei genes was used in this study. Raw milk artificially contaminated by different concentrations of Staph. aureus FRI 137 was employed in dairy processing resembling traditional raw milk cheese making. Samples of milk and curds were PCR-analysed after DNA extraction by targetting all the above genes. The pathogen was detected when the initial contamination was 10(4) CFU ml(-1) by amplification of nuc and seh genes. 10(5) and 10(7) CFU ml(-1) were needed when seg or sei and sec genes were targetted, respectively. Enrichment cultures from raw milk and curd samples proved to increase the detection limit of 1 log on average. CONCLUSIONS: The direct detection of the pathogen in the raw material and dairy intermediates of production can provide rapid results and highlight the presence of loads of Staph. aureus potentially representing the risk of intoxication. However, every target gene to be used in the analysis has to be studied in advance in a system similar to the real case in order to determine the level of contamination potentially predictable. SIGNIFICANCE AND IMPACT OF THE STUDY: The detection in real dairy systems of significant loads of Staph. aureus by multiple targets PCR can be more accurate.

PCR-DGGE fingerprints of microbial succession during a manufacture of traditional water buffalo mozzarella cheese.

AIMS: To monitor the process and the starter effectiveness recording a series of fingerprints of the microbial diversity occurring at different steps of mozzarella cheese manufacture and to investigate the involvement of the natural starter to the achievement of the final product. METHODS AND RESULTS: Samples of raw milk, natural whey culture (NWC) used as starter, curd after ripening and final product were collected during a mozzarella cheese manufacture. Total microbial DNA was directly extracted from the dairy samples as well as bulk colonies collected from the plates of appropriate culture media generally used for viable counts of mesophilic and thermophilic lactic acid bacteria (LAB) and used in polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) experiments. The analysis of the DGGE profiles showed a strong influence of the microflora of the NWC on the whole process because after the starter addition, the profile of all the dairy samples was identical to the one shown by the NWC. Simple indexes were calculated for the DGGE profiles to have an objective estimation of biodiversity and of technological importance of specific groups of organisms. LAB grown on Man Rogosa Sharp (MRS) and Rogosa agar at 30 degrees C showed high viable counts and the highest diversity in species indicating their importance in the cheese making, which had not been considered so far. Moreover, the NWC profiles were shown to be the most similar to the curd profile suggesting to be effective in manufacture. CONCLUSIONS: The PCR-DGGE analysis showed that in premium quality manufacture the NWC used as starter had a strong influence on the microflora responsible for process development. SIGNIFICANCE AND IMPACT OF THE STUDY: The molecular approach appeared to be valid as a tool to control process development, starter effectiveness and product identity as well as to rank cheese quality.

Isolation and technological properties of coagulase negative staphylococci from fermented sausages of Southern Italy.

The aims of this study were to characterize the population of Micrococcaceae in different types of fermented sausages of Southern Italy and to determine the technological properties of Staphylococcus strains in order to evaluate the suitability of selected strains as starter cultures in the processing of dry fermented pork sausages. Ninety-six strains were studied to evaluate nitrate reductase, proteolytic, lipolytic and antioxidant activities as well as growth ability at different temperatures, pH's and NaCl concentrations. All the strains were classified as Staphylococcus except for one isolate assigned to Kocuria spp. The species most often isolated were S. saprophyticus, S. xylosus and S. equorum, although they were not equally distributed within the different sausages. Other species isolated were, in descending order of abundance, S. succinus, S. warneri, S. lentus, S. vitulus, S. pasteuri, S. epidermidis, and S. haemolyticus. In general, the S. xylosus strains exhibited the best technological properties that would make them eligible as good starter cultures for fermented meat products. However, strains belonging to other species also showed good technological properties. Finally, all strains grew at 10, 15 and 20 °C, in the presence of 10% and 15% of NaCl and at pH 5.0 and 5.5. The results showed that it is possible to formulate a broad variety of staphylococcal starter cultures, adaptable to different technological conditions and sausage manufacture practices.

Selection of Lactobacillus strains from fermented sausages for their potential use as probiotics.

A rapid screening method was used to isolate potentially probiotic Lactobacillus strains from fermented sausages after enrichment in MRS broth at pH 2.5 followed by bile salt stressing (1% bile salts w/v). One hundred and fifty acid- and bile-resistant strains were selected, avoiding preliminary and time-consuming isolation steps. Strains were further characterized for survival at pH 2.5 for 3 h in phosphate-buffered saline and for growth in the presence of 0.3% bile salts with and without pre-exposure at low pH. Twenty-eight strains showed a survival >80% at pH 2.5 for 3 h; moreover, most of the strains were able to grow in the presence of 0.3% bile salts. Low pH and bile resistance was shown to be dependent on both the species, identified by phenotypic and molecular methods, and the strain tested. This is the first report on the direct selection of potentially probiotic lactobacilli from dry fermented sausages. Technologically interesting strains may be used in the future as probiotic starter cultures for novel fermented sausage manufacture.

Comparison of statistical methods for identification of Streptococcus thermophilus, Enterococcus faecalis, and Enterococcus faecium from randomly amplified polymorphic DNA patterns.

Thermophilic streptococci play an important role in the manufacture of many European cheeses, and a rapid and reliable method for their identification is needed. Randomly amplified polymorphic DNA (RAPD) PCR (RAPD-PCR) with two different primers coupled to hierarchical cluster analysis has proven to be a powerful tool for the classification and typing of Streptococcus thermophilus, Enterococcus faecium, and Enterococcus faecalis (G. Moschetti, G. Blaiotta, M. Aponte, P. Catzeddu, F. Villani, P. Deiana, and S. Coppola, J. Appl. Microbiol. 85:25-36, 1998). In order to develop a fast and inexpensive method for the identification of thermophilic streptococci, RAPD-PCR patterns were generated with a single primer (XD9), and the results were analyzed using artificial neural networks (Multilayer Perceptron, Radial Basis Function network, and Bayesian network) and multivariate statistical techniques (cluster analysis, linear discriminant analysis, and classification trees). Cluster analysis allowed the identification of S. thermophilus but not of enterococci. A Bayesian network proved to be more effective than a Multilayer Perceptron or a Radial Basis Function network for the identification of S. thermophilus, E. faecium, and E. faecalis using simplified RAPD-PCR patterns (obtained by summing the bands in selected areas of the patterns). The Bayesian network also significantly outperformed two multivariate statistical techniques (linear discriminant analysis and classification trees) and proved to be less sensitive to the size of the training set and more robust in the response to patterns belonging to unknown species.

Molecular evaluation of microbial diversity occurring in different types of Mozzarella cheese.

AIMS: The microbial community of different types of unripened Pasta Filata cheese was investigated by culture-independent methods with the aim of rapidly achieving knowledge about cheese microbiota and discriminating traditional and industrial cheeses. METHODS AND RESULTS: The microbial DNA extracted directly from the samples was used as a template in PCR experiments to amplify the 16S-23S rDNA spacer region and the V3 region of the 16S rDNA. Conventional electrophoresis of the amplified spacers allowed known classes of these DNA fragments belonging to genera and species of lactic acid bacteria to be distinguished. Denaturing gradient gel electrophoresis analysis of V3 amplicons was supported by reference cultures of LAB used as markers. CONCLUSION: Both molecular approaches furnished the expected information about microbial diversity and were quite valid for discriminating industrial, semi-artisanal or traditional cheeses, characterized by increasingly complex DNA profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: Both methods could be used for legal purposes when products obtained through prescribed manufacturing regulations are to be analysed.