Giant reed growth and effects on soil biological fertility in assisted phytoremediation of an industrial polluted soil.

Phytoremediation is a cost-effective "green technology" that uses plants to improve the soil properties of polluted sites, preventing the dispersion of pollutants and reducing the mobility of potentially toxic elements (PTEs) through their adsorption and accumulation by roots or precipitation within the root zone. Being highly tolerant to pollutants and other abiotic stresses, giant reed (Arundo donax L.) is a suitable biomass crop for phytoremediation of contaminated soils. We report the results of a two-year open-air lysimeter study aimed at assessing the adaptability of giant reed to grow on industrial substrates polluted by Pb and Zn and at testing commercial humic acids from leonardite as improvers of plant performance. We evaluated giant reed potential for: 1) biomass production for energy or biomaterial recovery; 2) PTE phytoextraction and 3) soil fertility restoration. Chemical fertility was monitored by measuring soil C while soil biological fertility was estimated by quantifying the abundance of bacterial functional genes regulating nitrogen fixation (nifH) and nitrification (amoA). Giant reed above-ground growth on the polluted soils was slightly lower (-16%) than on a non-polluted soil, with a preferential storage of biomass in the rhizome acting as a survival strategy in limiting growing conditions. Humic acids improved plant stress tolerance and production levels. As aerial biomass (shoots) did not accumulate PTEs, the plant in question can be used for bioenergy or biopolymer production. In contrast, below-ground biomass (rhizomes) accumulated PTEs, and can thus be harvested and removed from soil to improve phytoremediation protocols and also used as industrial biofuel. Giant reed growth increased the abundance of N-cycling bacteria and soil C in the rhizospheric soil, as well as reduced soil Pb and Zn EDTA extractable fraction.

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.

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.

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.

Effect of proteolytic starter cultures as leavening agents of pizza dough.

Lactic acid bacteria (LAB) and yeasts were selected on the basis of in vitro proteolytic activity against wheat gluten protein and then assayed as leavening agents for pizza dough. Trials were carried out to compare a proteolytic starter (Prt(+)), consisting of Lactobacillus sakei T56, Weissella paramesenteroides A51 and Candida krusei G271, and a non-proteolytic starter (Prt(-)), consisting of Lb. sakei T58, W. paramesenteroides A58 and Saccharomyces cerevisiae T22. The proteolytic activity of the starter cultures was monitored immediately after mixing of the dough and throughout the fermentation process. The proteolytic activity was assessed by analysing the salt-soluble protein (SSP) and the dioxane-soluble protein (DSP) fractions of the pizza dough by discontinuous SDS-PAGE. Only the Prt(+) starter exhibited considerable qualitative and quantitative changes in the electrophoretic patterns of the protein fractions extracted. After the fermentation, the Prt(+) and Prt(-) doughs were tested to evaluate the influence of the proteolytic activity on the mechanical properties of the dough before and after baking. Indications emerged suggesting an influence of the proteolytic activity on the viscoelasticity of pizza dough. The pizza dough with Prt(+) strains showed an increase in viscous properties during the fermentation as compared with the Prt(-) dough. Moreover, an increase in the firmness of the crumb was observed in Prt(+) baked pizza dough.

Differential viable count of mixed starter cultures of lactic acid bacteria in doughs by using modified Chalmers medium.

The modified Chalmers medium appeared as quite suitable for counting a mixed population consisting of different species of lactic acid bacteria used as starter in breadmaking. Selected strains of Lactobacillus plantarum, Leuconostoc mesenteroides subsp. mesenteroides, Lactobacillus sanfranciscensis, Enterococcus faecalis together with Saccharomyces cerevisiae could be easily differentiated and counted with an acceptable recovery in comparison with reference media.

Detection and characterization of a bacteriocin, garviecin L1-5, produced by Lactococcus garvieae isolated from raw cow's milk.

AIMS: The identification of a bacteriocin-producing lactococcal strain isolated from raw cow's milk is reported, along with production conditions, physical and chemical properties, and mode of action of the bacteriocin. METHODS AND RESULTS: On the basis of resistance to clindamycin, species-specific PCR and amplification of the 16S-23S rDNA spacer region, the strain was identified as Lactococcus garvieae. Its bacteriocin, designated garviecin L1-5, was bactericidal against closely related species and strains of species from different genera, including Listeria monocytogenes and Clostridium spp. Garviecin L1-5 was shown to be proteinaceous by protease inactivation and was unaffected by heat treatments, also at low pH values. When amplifying known lactococcal bacteriocin genes using DNA from strain L1-5 as template, no amplification products were observed on the agarose gel. The molecular weight of garviecin L1-5 was about 2.5 kDa. As far as is known, no bacteriocins have been detected from Lactococcus garvieae. CONCLUSION: The general properties of garviecin L1-5 are characteristic of the low-molecular-weight bactericidal peptide group. SIGNIFICANCE AND IMPACT OF THE STUDY: The survey of micro-organisms for novel antimicrobial substances provided valuable information on their physiology, ecology and practical application.

Effect of leavening microflora on pizza dough properties.

Fourteen different starter cultures containing one strain of Saccharomyces cerevisiae with and without individual or combinations of lactic acid bacteria (Lactobacillus plantarum, Lact. sanfrancisco, Enterococcus faecium, Leuconostoc mesenteroides) were employed to investigate the role of leavening microflora on the properties of pizza doughs. Microbiological, chemical and physical characteristics of doughs prepared with the same flour and under the same processing conditions were determined. Leavening times and acidification properties depended on the microbial association used. The proportions of lactic and acetic acid produced by lactic acid bacteria were consistent with the metabolic properties of the strains employed. The bacteria/yeast ratios arising from microbial counts at the end of the leavening process were always lower in comparison to sour- or bread-doughs. The size of the yeast population did not change much, while bacteria showed from one to four duplications. Rheologically, the fermented doughs could only be significantly distinguished from the control dough with regard to the elastic modulus. Principal Component Analysis was applied to the acidimetric data. The scattergram of the two principal components effectively discriminated 13 of the 14 pizza dough types.

Behaviour of Listeria monocytogenes during the traditional manufacture of water-buffalo Mozzarella cheese.

The behaviour of a four-strains mixture of Listeria monocytogenes (strains Scott A, V7, OH and Cal) during the traditional manufacture of water-buffalo Mozzarella cheese was investigated at two levels of inoculation: ca 10(5) and 10(3) cfu ml-1 of vat milk. No significant change in Listeria counts was observed during the curd ripening (4.0-4.5 h), at the end of which the pH ranged between 4.83 and 4.91. A decrease of about 2 log was observed after stretching of the curd in hot water (95 degrees C), followed by complete elimination of Listeria after 48 and 24 h of storage of the final cheese in the conditioning liquid (skim water resulting from the stretching, pH ca 4.0) with initial high and low contamination of the cheese milk respectively. Results also indicated that a 1.7 log reduction of L. monocytogenes could be achieved during the preparation of the natural whey culture utilized as starter in cheesemaking.

Antilisterial activity of thermophilin 347, a bacteriocin produced by Streptococcus thermophilus.

Streptococcus thermophilus 347 isolated from yogurt produces a bacteriocin referred as thermophilin 347. The bacteriocin was evidenced in the neutralized, filtered and catalase treated culture supernatant fluid of the producer strain. After partial purification, thermophilin 347 exhibited a bactericidal effect against Listeria monocytogenes and several closely related lactic acid bacteria. The activity of thermophilin 347 was lost after protease treatment but was maintained after heating at 100 degrees C for 1 h; after autoclaving at 121 degrees C for 15 min the activity was reduced by 50%. SDS-PAGE of partially purified thermophilin 347 was used to detect bacteriocin activity corresponding to an apparent molecular mass between 2.5 and 6.2 KDa.

Genotyping of Streptococcus thermophilus evidenced by restriction analysis of ribosomal DNA.

Twenty strains of Streptococcus thermophilus were classified into five different types characterized by ribotyping after DNA digestion with HindIII and hybridization with cDNA from 16S-23S rDNA of Escherichia coli. Ribotyping after digestion with HaeIII and XhoI enabled the same strains to be gathered into three groups as a consequence of the reduced influence of the flanking sequences within the analysis of rRNA operons. Amplified rDNA restriction analysis clearly confirmed the discrimination of these three different ribotypes, producing evidence to suggest that subtaxa are to be recognized within this species.

Enterocin 226NWC, a bacteriocin produced by Enterococcus faecalis 226, active against Listeria monocytogenes.

Enterococcus faecalis 226, isolated from natural whey cultures utilized as starters in the manufacture of mozzarella cheese from water-buffalo milk, produces a bacteriocin designated enterocin 226NWC. The bacteriocin was isolated from culture supernatant fluids of the producer strain and was active against strains of the same species and Listeria monocytogenes, but not against useful lactic acid bacteria. Enterocin 226NWC is a protein with an apparent molecular weight of about 5800; it is relatively heat-stable and has a bactericidal mode of action. Listeria monocytogenes, growing in the presence of the enterocin 226NWC producer strain in broth and in reconstituted skim milk, was inhibited.

Conjugal transfer of plasmid-borne bacteriocin production in Enterococcus faecalis 226 NWC.

Enterococcus faecalis 226 NWC, isolated from natural whey cultures utilized as starter in water-buffalo Mozzarella cheese manufacture, produces a bacteriocin, designated Enterocin 226 NWC, which is inhibitory to Listeria monocytogenes. Plasmid analysis of E. faecalis 226 NWC showed a single 5.2-kb plasmid, pEF226. In conjugation experiments, pEF226 was transferred into a plasmid-free strain of E. faecalis JH2-2. The transfer required direct cell-to-cell contact and was not inhibited by DNase. The identity of conjugation was confirmed by digestion with SmaI restriction endonuclease and subsequent pulsed-field gel electrophoresis (PFGE) of the genomic DNA of E. faecalis 226, E. faecalis JH2-2 and of the isolates after the mating. The data indicate that the ability of E. faecalis 226 NWC to produce the bacteriocin is linked to the 5.2-kb conjugative plasmid pEF226.