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Extensional deformations represent an effective stimulus to explore the rich rheological response of branched polymers and elastomers, enabling the design of polymers with specific molecular structure. However, probing the polymer behavior at large deformations is often limited by the experimental devices. We here present an alternative use of the Sentmanat Extensional Rheometer (SER) that allows Hencky strain units much larger than the maximum value achievable, ∼3.6. The proposed procedure consists of an oblique positioning of the sample in the measuring area. If a small inclination of the sample is used, the departure from the ideal uniaxial flow is negligible at Hencky strains

Dynamic mechanical oscillatory shear measurements with torsional rectangular geometry are widely carried out in order to determine the mechanical properties of soft solid materials in a quick and practical way. This technique has the advantage of avoiding slip effects, thus being particularly attractive for testing stiff elastomers such as vulcanized rubber compounds. However, one of its drawbacks is the clamping system required to keep the specimen edges in place. Since it imposes a constraint to warping deformations (i.e., out-of-plane cross-section distortions about the torsional axis), a certain increase of dynamic moduli with respect to their values in simple shear is observed and considered as an experimental artifact (i.e., de Saint-Venant's assumption of primary torsion of the specimen is no longer valid). The increase of dynamic moduli in torsion depends on the specimen's cross-section geometry and relative dimensions. We test here the capability of different torsion models to describe the constrained warping effect for an industrial rubber with rectangular specimen cross section using a wide range of different geometric parameters (length-to-width p ratio and width-to-thickness u ratio). We compare two theoretical models (Vlasov's model and Timoshenko's approach) and a phenomenological model (based on finite element simulations by Diani and Gilormini) with our experimental data set. We propose a slight modification of Vlasov's model to obtain realistic predictions of torsion over a wider parameter range. It is shown to be particularly useful in soft rubber testing, and the limitations in the choice of specimen geometry to obtain sufficient torque signal are overcome.

We report upon the characterization of the steady-state shear stresses and first normal stress differences as a function of shear rate using mechanical rheometry (both with a standard cone and plate and with a cone partitioned plate) and optical rheometry (with a flow-birefringence setup) of an entangled solution of asymmetric exact combs. The combs are polybutadienes (1,4-addition) consisting of an H-skeleton with an additional off-center branch on the backbone. We chose to investigate a solution in order to obtain reliable nonlinear shear data in overlapping dynamic regions with the two different techniques. The transient measurements obtained by cone partitioned plate indicated the appearance of overshoots in both the shear stress and the first normal stress difference during start-up shear flow. Interestingly, the overshoots in the start-up normal stress difference started to occur only at rates above the inverse stretch time of the backbone, when the stretch time of the backbone was estimated in analogy with linear chains including the effects of dynamic dilution of the branches but neglecting the effects of branch point friction, in excellent agreement with the situation for linear polymers. Flow-birefringence measurements were performed in a Couette geometry, and the extracted steady-state shear and first normal stress differences were found to agree well with the mechanical data, but were limited to relatively low rates below the inverse stretch time of the backbone. Finally, the steady-state properties were found to be in good agreement with model predictions based on a nonlinear multimode tube model developed for linear polymers when the branches are treated as solvent.

We present a simple new analytical method for educing the materials' linear viscoelastic properties, over the widest range of experimentally accessible frequencies, from a simple step-strain measurement, without the need of preconceived models nor the idealization of real measurements. This is achieved by evaluating the Fourier transforms of raw experimental data describing both the time-dependent stress and strain functions. The novel method has been implemented into an open access executable "i-Rheo," enabling its use to a broad scientific community. The effectiveness of the new rheological tool has been corroborated by direct comparison with conventional linear oscillatory measurements for a series of complex materials as diverse as a monodisperse linear polymer melt, a bimodal blend of linear polymer melts, an industrial styrene-butadiene rubber, an aqueous gelatin solution at the gel point and a highly concentrated suspension of colloidal particles. The broadband nature of the new method and its general validity open the route to a deeper understanding of the material's rheological behavior in a variety of systems. (C) 2016 The Society of Rheology.

Fiber spinning experiments are conducted with a capillary rheometer and a Rheotens tester on linear styrene-isoprene-styrene copolymer samples by varying extrusion temperature and drawdown velocity in a wide range of values, also covering the occurrence of instability phenomena. Tensile stress is measured during the experiences, and the experimental time series are then analyzed by means of a new methodology. The proposed approach is based on Neural Network modeling of the time series, coupled with Principal Component Analysis postprocessing of the results. The methodology is able to identify and quantify the effects of process condition on the dynamical behavior of the system. POLYM. ENG. SCI., 2013. © 2012 Society of Plastics Engineers

We probed the viscoelastic behavior of polydisperse star/linear polymer blends with varying composition and average arm length. The linear viscoelastic behavior has been investigated over a wide range of frequencies using both oscillatory and long creep/recovery experiments. A method was developed for splitting the molar mass distributions of the blends into the contributions of different architectures. These results were used to implement a time-marching algorithm based on a parameter-free tube model for predicting the linear viscoelastic behavior. The model predictions were successfully validated with the experimental data, opening the route for designing blends with desired rheological properties.

A set of degenerate PCR primers was designed and used to amplify and sequence about 75% of the catalase ( kat ) gene from each of 49 staphylococcal strains. In some strains of Staphylococcus xylosus , S. saprophyticus , and S. equorum , two catalase genes, kat A and kat B , were found. A phylogenetic tree was generated and showed diversities among 66 partial (about 900-bp) staphylococcal kat nucleotide sequences (including 17 sequences found in GenBank) representing 26 different species. The topology of this tree showed a distribution of staphylococcal species similar, but not identical, to those reported previously based on 16S rRNA, hsp 60 , sod A , rpo B , tuf , and gap genes. The kat gene sequences were less conserved than those of 16S rRNA, rpo B , hsp 60 , and tuf genes and slightly more conserved than those of the gap gene. Therefore, kat gene sequence analysis may provide an additional marker for inferring phylogenetic relationships of staphylococci. Moreover, the discrete nucleotide polymorphism revealed in this gene could be exploited for rapid, low-cost identification of staphylococcal species through PCR-restriction fragment length polymorphism (RFLP) analysis. In this study, a PCR-RFLP assay performed by using only the TaqI restriction enzyme was successfully developed for rapid unequivocal identification/differentiation, at species and subspecies levels, of coagulase-positive staphylococci (CPS). The assay was validated by testing the DNA from 100 staphylococcal strains, including reference and wild CPS strains isolated from different environments. This reliable, rapid, and low-cost approach (requiring about 6 h from DNA isolation to the achievement of results and S. delphini and S. pseudintermedius CPS species.

New data of shear startup on branched styrene-butadiene random (SBR) copolymers are reported, where the novelty consists in repeating the startup run after different rest times at zero stress. Here, the aim is one of exploring the “damage” introduced by the first run, as well as the subsequent recovery, if any, upon waiting increasingly long times. Differently from a linear sample, our branched melts show multiple peaks during the first run, as previously reported by Bacchelli [Kautschuk Gummi Kunststoffe 61, 188–191 (2008)] for similar SBR samples, and, more recently, by Snijkers et al. [ACS Macro Lett. 2, 601–604 (2013)] for a well-characterized comblike polystyrene melt. The repeated runs show an intriguing novel feature with respect to the case of linear polymers, namely, the first peak goes up initially, instead of down. The second peak goes down and seemingly recovers only after an extremely long time, longer than the largest relaxation time practically accessible to linear viscoelasticity, the latter not reaching the terminal behavior. All such features of nonlinear viscoelasticity of highly branched polymers are interpreted by using a simple theory inspired by the well-known pompom model.

We investigate the dynamic response of industrial rubbers (styrene-butadiene random copolymers, SBR) in torsion and compare against common small amplitude oscillatory shear measurements by using a torsion rectangular fixture, a modified torsion cylindrical fixture, and a conventional parallel plate fixture, respectively, in two different rheometers (ARES 2kFRTN1 from TA Instruments, USA and MCR 702 from Anton Paar-Physica, Austria). The effects of specimen geometry (length-to-width aspect ratio) on storage modulus and level of clamping are investigated. For cylindrical specimens undergoing torsional deformation, we find that geometry and clamping barely affect the shear moduli, and the measurements essentially coincide with those using parallel plates. In contrast, a clear dependence of the storage modulus on the aspect ratio is detected for specimens having rectangular cross section. The empirical correction used routinely in this test is based on geometrical factors and can account for clamping effects, but works only for aspect ratios above a threshold value of 1.4. By employing a finite element analysis, we perform a parametric study of the effects of the aspect ratio in the cross-sectional stress distribution and the linear viscoelastic torsional response. We propose a new, improved empirical equation for obtaining accurate moduli values in torsion at different aspect ratios, whose general validity is demonstrated in both rheometers. These results should provide a guideline for measurements with different elastomers, for which comparison with dynamic oscillatory tests may not be possible due to wall slip issues.

We investigated the linear melt viscoelasticity and the crystallization kinetics of a series of model poly(ethylene oxide) stars with different functionalities (f=4-32 arms) and moderately entangled arms (their molecular masses ranging from 5.5 to 12 kg/mol). The limited data in the homogeneous state indicated that the zero-shear viscosity eta(0) was adequately described by the Milner-McLeish model for functionalities f < 32, where the core effect is insignificant; a similar behavior was observed for the recoverable compliance J(e)(0) which depended on both the molecular weight and the number of the arms. Below the melting point, the isothermal crystallization was measured with differential scanning calorimetry and rheology, and analyzed in terms of the Avrami theory, expanding over a wide range of temperatures. The results were supported by additional polarizing optical microscopy data and indicated a slower crystallization kinetics of the stars compared to their linear analogues. They showed a strong dependence of the crystallization rate on the arm molecular weight, whereas the available experimental evidence is suggestive of some functionality dependence as well. (c) 2007 The Society of Rheology. Journal of Rheology

Sequences of the lacSZ operon of 29 Streptococcus thermophilus strains from different dairy products were determined. Differences in sequence among the strains were detected within LacS more often than in the LacZ gene. The sequences were aligned and compared and it was possible to gather the strains into three groups of similarity on the basis of the LacS gene sequence. The dairy environment of origin did not seem to be related to the lacSZ operon sequence and thus to the similarity shown. Nucleotide variability was investigated and a total of 139 nucleotide changes were found in the LacS gene while 40 nucleotide changes were found in the sequences of the LacZ gene. Moreover, the influence of the nucleotide changes on the amino acid sequence of the LacS transporter and of the β-galactosidase enzyme were discussed. Sequence variability within the region upstream from the LacS gene was used to develop group-specific PCR systems capable of distinguishing S. thermophilus at the strain level. A strain-specific primer set was designed allowing the specific detection of 11 out of 29 strains of S. thermophilus. Moreover, LacS-PCR-SSCP analysis of the 29 strains provided 2 different profiles, whereas 4 strain-specific profiles were detected by LacS-PCR-DGGE, indicating the potential to use these techniques for profiling and monitoring population of strains of S. thermophilus in food products. The results are discussed with reference to the potential of these PCR methods for ascertaining strain dominance and starter fitness in dairy processes.

This study investigates the coupled effects of mild shear flow and temperature on the crystallization behavior of two thermoplastic polymers, namely, an isotactic polypropylene and an isotactic poly(1-butene). Rheological experiments are used to measure the crystallization induction time under isothermal, steady shear flow conditions. The experimental results clearly show the effects of the degree of undercooling on flow-induced crystallization (FIC). As temperature decreases, the corresponding increase in chain orientation at a given shear rate leads to an absolutely faster crystallization. At the same time, however, a temperature decrease makes the flow-induced driving force to crystallization relatively less influent with respect to the intrinsic kinetics. A FIC model based on the Doi–Edwards micro-rheological theory is shown to successfully describe the quantitative details of the observed experimental behavior.

A phenotypic and genotypic characterization of 84 Oenococcus oeni isolates from Italian wines of different oenological areas was carried out. Numerical analysis of fatty acid profiles grouped the isolates into two clusters at low level of similarity (63%), the minor cluster containing seven isolates besides the type and the reference strains. Forthy-eight O. oeni isolates, representative of the two clusters, showed no differences in their metabolic properties (heterolactic fermentation pattern, citrate degradation capability and formation of some secondary metabolites). Moreover, the analysis of species-specific randomly amplified polymorphic DNA and 16S–23S rDNA intergenic spacer region polymorphism as well as the sequence-specific separation of V3 region from 16S rDNA by denaturing gradient gel electrophoresis demonstrated a substantial homogeneity among the isolates. On the basis of Apa I Pulsed Field Gel Electrophoresis (PFGE) restriction patterns, the 84 isolates were grouped into five different clusters at 70% similarity, but no correlation with the phenotypic groups could be demonstrated. However, by combining phenotypic and genotypic data, the 84 O. oeni isolates grouped into eight phenotypic–genotypic combined profiles and a relationship between the origin of the isolates and their combined profile became evident, so that a sort of strain specificity can be envisaged for each wine-producing area.

G. MAURIELLO, L. MOIO, G. MOSCHETTI, P. PIOMBINO, F. ADDEO AND S. COPPOLA. 2001. Aims: Seventy-eight strains of lactic acid bacteria belonging to five genera and showing six different phenotype combinations of Lac (lactose fermentation), Prt (proteolytic activity) and Cit (citrate degradation) characters were investigated for their main flavouring properties with the aim to detect variability among and within the groups. Methods and Results: High resolution gas chromatography–mass spectrometry analysis of neutral volatile compounds produced in whey showed that, considering both neo-formation compounds and substances quantified in the whey cultures at different concentrations in comparison to the extract from sterile whey, the groups of lactococci, enterococci, thermophilic streptococci and mesophilic lactobacilli produced a higher number of volatiles than thermophilic lactobacilli and leuconostocs. Applying principal component analysis (PCA) to the results, enterococci, mesophilic lactobacilli and thermophilic streptococci showed a broad diversity, while lactococci included rather similar strains as well as strains with special flavouring properties. Applying PCA to thermophilic streptococci and enterococci, to lactococci and enterococci, to lactococci and thermophilic streptococci, or to mesophilic and thermophilic lactobacilli, the strains gathered consistently with their systematic position. Conclusions: The study evidenced strains producing some volatile compounds responsible for food flavouring. Flavouring properties were variable among the systematic groups and in some cases different within the same bacterial group. Significance and Impact of the Study: The potential of the findings is discussed with reference to the development of flavouring adjuncts for the dairy industry.

Lactic acid bacteria (LAB) play an important role in food fermentation, as the products obtained with their aid are characterized by hygienic safety, storage stability and attractive sensory properties. A major aim of the research in this field is the selection of LAB strains that could be used. Hence it is very important to be able to apply a reliable method to distinguish a particular strain specifically and unambiguously, which allows studies of population dynamics of mixed cultures and monitoring starter strains during fermentation (Ramos & Harlander, 1990).Molecular methods are a powerful alternative to the traditional differentiation of bacteria. A highly reproducible method for characterizing and distinguishing closely related strains, is represented by REA-PFGE (restriction endonuclease analysis by pulsed-field gel electrophoresis) performed by infrequently cutting endonucleases. Genetic differentiation of strains in several species of LAB has been successfully performed by this technique (Moschetti et al. 1997; Villani et al. 1997) obtaining very clear and reproducible restriction patterns (Moschetti et al. 1998).In this study selected inoculated strains (lactococci or lactobacilli) were monitored by REA-PFGE during the whole process of water-buffalo ‘cacioricotta’ cheese-making. This product is a typical and traditional cheese in southern Italy produced from cow, goat, ewe or water-buffalo milk. The original technology of this preparation permits the recovery of whey proteins due to the high heat treatment of whole milk employed, allowing interesting yields to be achieved in terms of cheese. The use of starters is not common in traditional technology but low acid protection of the final product suggested the use of LAB as starter (Emaldi et al. 1987).

A polyphasic PCR-DGGE approach was used to describe the microbial population occurring in natural whey cultures (NWCs) for water-buffalo Mozzarella cheese production. Total microbial community was assessed without cultivation by analyzing DNA directly extracted from the original samples of NWC. In addition, DNA extracted from bulks of cells formed by harvesting colonies from the serial dilution agar plates of a variety of culture media was used to profile the "cultivable" community. The 16S rDNA V3 region was amplified using DNA from NWC as well as DNA from bulks as templates and the amplicons were separated by DGGE. The microbial entities occurring in NWCs were identified by partial 16S rDNA sequencing of DGGE bands: four lactic acid bacteria (LAB) closest relative of Streptococcus thermophilus, Lactococcus lactis, Lactobacillus delbrueckii and Lactobacillus crispatus were revealed by the analysis of DNA directly extracted from NWC while two other LAB, Lactobacillus fermentum and Enterococcus faecalis, were identified by analyzing DNA from the cultivable community. The developed PCR-DGGE analysis of the "cultivable" community showed good potential in evaluating microbial diversity of a dairy environment: it usefully highlighted the bias introduced by selective amplification when compared to the analysis of the total community from NWC and allowed suitability of media and growth conditions to be evaluated. Moreover, it could be used to complete the culture independent study of microbial diversity to give information on concentration ratios among species occurring in a particular environment and can be proposed for rapid identification of dominant microorganisms in alternative to traditional tools.

Sixty-seven lactococcal strains arising from dairy habitat were screened for the presence of the sucrose 6-phosphate hydrolase gene by polymerase chain reaction. Of the strains tested, 35.8% were able to ferment sucrose as well as to harbour the sucrose-6-phosphate hydrolase gene, even though they were unable to produce nisin as well as to show the nisin structural gene. After pulsed-field gel electrophoresis and hybridisation all Suc+Nis− strains exhibited physical linkage between sacA gene and the left end of lactococcal transposons (Tn5276 or Tn5301) without linkage to nisin genes. However, we were unable to transfer the sacA gene as well as to detect Suc− derivatives from Suc+Nis− strains after conjugation and curing experiments.

Staphylococcus xylosus 1E, isolated from Italian sausages, produced a substance with antagonistic activity against Listeria monocytogenes and other gram-positive bacteria during growth on a solid medium. Inhibitory activity in cell-free supernatant fluids of cultures in various broths was unsuccessfully researched. An active preparation of the inhibitory substance was obtained from the cells of the producer strain by desorption with 9moll −1 urea. The inhibitor was not affected by autoclaving (121°C for 15 min), whereas 50% activity remained after pronase and esterase treatment. SDS-PAGE allowed a diffuse smear of stainable material with inhibitory activity to be detected. The 1E inhibitor exhibited a bactericidal mode of action through adsorption to the cells and leakage of nucleic acids. The inhibition of L. monocytogenes by S. xylosus 1E was also observed in Naples-type sausages. After 21 days of maturation, in salami in which L. monocytogenes was challenged with S. xylosus 1E, the viable counts of the pathogen were reduced by approximately 2 log cycles, whereas 0.5 log reduction occurred in sausages with the pathogen alone. No L. monocytogenes were recovered after 75 days in sausages inoculated with S. xylosus 1E, while the pathogen was still present at this time in control sausages.

Isolated microperfused rabbit renal proximal tubule S2 segments, if incubated in conventional substrate containing HCO3- Ringer solution, exhibit lower cell membrane potentials (Vb) and elevated intracellular Na+ concentrations ([Na]i) compared to rat tubules in vivo. Assuming that these and other differences reflect insufficient metabolic and/or hormonal stimulation of the cells, we have used microelectrode techniques to test whether improving substrate supply and applying norepinephrine (NE, to compensate for the missing nerve supply) reverts Vb and [Na]i to values observed in vivo. Application of D-glucose (5.5 mmol/l) and additional application of pyruvate, lactate, or L-alanine (each 10 mmol/l), or bathing the tubules in Dulbecco's modified Eagle's tissue culture medium (DMEM) significantly increased Vb and, whenever tested, reduced [Na]i as compared to substrate-free or D-glucose-containing control solution and these effects could be prevented - as tested in the case of pyruvate - by inhibition of the Na/K pump with ouabain. However, high concentrations of acetate, beta-hydroxybutyrate, or L-glutamine had no significant effect. The largest effect was obtained with joint application of DMEM and NE (10 micromol/l) which increased Vb from -42.8 +/- 1.3 mV (SEM) to -55.3 +/- 2.5 mV (n = 11). Interestingly we noticed that under the latter conditions the Vb response to bath application of 1 mmol/l amiloride virtually disappeared, i.e. it changed from a depolarization of +14.6 +/- 1.4 mV (in D-glucose Ringer solution) to +0.6 +/- 0.7 mV (in DMEM plus NE) (n = 8), with some tubules showing even a small hyperpolarization. The latter implies partial restoration of the in vivo behaviour, since in experiments on rat proximal tubules in vivo amiloride regularly hyperpolarized the cells (by -3.4 +/- 0.76 mV, n = 5). Obviously under conventional in vitro conditions an amiloride-inhibitable K+ conductance is activated which is inactive in vivo and also inactivates under improved conditions in vitro. In agreement with observations reported in the subsequent publication our results demonstrate that isolated proximal tubules undergo functional alterations which may be largely prevented by improved metabolic and stimulatory incubation conditions.

Summary Three different approaches (whole-cell protein profiles, DNA fingerprinting combined with pulsed-field gel electrophoresis and analysis of rDNA genes) were used to characterize thirty-one strains of Lactobacillus delbrueckii subsp. bulgaricus from different dairy products, and three type strains belonging to L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. lactis and L. delbrueckii subsp. bulgaricus. Moreover, the number and different forms of rrn operons in L. delbrueckii and its subspecies were defined. At the strain typing level, NotI macrorestriction analysis permitted grouping of the 32 strains of L. delbrueckii subsp. bulgaricus into 23 restriction patterns, providing a high degree of discriminatory power. Among whole-cell protein profiles, PCR analysis of rDNA genes and ribotyping, the latter method seemed to be the most reliable approach to characterizing the subspecies belonging to L. delbrueckii. Ribotyping combined with enzymes such as HindIII and EcoRI showed that at least six rrn operons were present in L. delbrueckii and its subspecies; two forms of rrn operons were present in the subspecies lactis and bulgaricus and four forms were present in the subspecies delbrueckii.

Admiral Alfred von Tirpitz’s naval programme reveals a number of underlying features which are unique aspects of the political culture of the Kaisereich; both in the domestic and international contexts. The naval arms-race against Great Britain was an event that resulted from the interaction of a number of goals that Tirpitz had initially envisaged for Germany and Kaiser William II. Yet, the pursuit of an ever-greater navy fleet was by no means a monopoly of the Germans but, on the contrary, it was a common trait of every major European Power of the time, from Britain to Austria, to Russia. Nevertheless, the scale to which Tirpitz went deserves a particular emphasis in that it was not only a military programme, but rather, its aims were directed towards the achievement ofobjectives in a number of fields: economic growth, domestic socio-political stability, and German Imperialism through Weltpolitik, which reflected the contemporary political issues present within the Kaiserreich. These three aspects will be explored with further emphasis on the structural aspectsof the Wilhelmine system of government together with the individuals that were involved in German policymaking at the time. It will therefore be possible to observe how these various features were crucial in determining Germany’s political system. This, in turn, allowed Tirpitz’s radical and militaristic plan to take place. The Tirpitz Plan reveals its objectives as being not solely restricted to the military-naval domain. Furthermore, one will see how the Tirpitz plan could have taken place only in Germany during the Kaiserreich thanks to the uniquely ambiguous administrative structures Germany possessed.

On the mechanism of bicarbonate exit from renal proximal tubular cells. We compare here the results of electrophysiological measurements on proximal tubular cells performed on rat kidney in vivo and on isolated rabbit and rat tubules in vitro . Based on different effects of carbonic anhydrase inhibitors in the in vivo and in vitro preparation, we conclude that NaHCO 3 cotransport across the basolateral cell membrane functions as Na + -CO 3 2 − -HCO 3 − cotransport in vivo , but as Na + -HCO 3 − -HCO 3 − cotransport in the classical in vitro preparation. The former, but not the latter, transport mode is characterized by generation of local disequilibrium pH / CO 3 2 − concentrations that oppose fluxes if membrane-bound carbonic anhydrase is inhibited. In support of this conclusion, we find that overall transport functions with a HCO 3 − to Na + stoichiometry of 3:1 in vivo (since each transported CO 3 2 − eventually generates 2 HCO 3 − ions), but 2:1 in vitro . This has been deduced from various measurements, among them super-Nernstian and reverse Nernstian, potential responses to changing ion concentrations which are characteristic of obligatorily coupled cation-anion cotransporters, but are not known in classical electrochemistry. The different transport modes in vivo and in vitro suggest that isolated proximal tubules have functional deficits compared to proximal tubules in vivo .

In the present experiments we have made a new attempt to characterize the ion transport properties of H(+)-secreting cells of the gastric mucosa using electrophysiological techniques. Individual gastric glands of bullfrog fundus mucosa were manually dissected, mounted in holding pipettes and superfused with various test solutions while individual cells were punctured with conventional or H(+)-sensitive double-barrelled microelectrodes. All measurements were performed in the resting state (0.1 mmol/l cimetidine). In HCO3(-)-containing control Ringer solution the cell membrane potential (Vb) averaged -45.6 +/- 0.9 mV (+/- SEM, n = 54). From the fast initial Vb responses to changing bath K+, Na+, Cl- or HCO3- concentrations we deduced that the basolateral cell membrane contains conductances for K+, Na+, and Cl- but not for HCO3-, and that a Na(+)-HCO3- cotransporter is not present. The K+ conductance was inhibited by Ba2+ (3 mmol/l), but the Cl- conductance was not inhibited by 4,4' diisothiocyanato-stilbene-2,2' disulphonic acid (DIDS, 0.3 mmol/l), nor selectively inhibited by 5-nitro-2-(3)- phenylpropyl-aminobenzoate (NPPB, 10 mumol/l). In a great number of cells the Vb response to Cl- substitution revealed two components: an initial spiking depolarization which reflected conductive Cl- efflux and a secondary slow hyperpolarization, the origin of which was not immediately evident. Since the latter response could be mimicked by CO2-free perfusion, strongly depressed by Ba2+ and eliminated by DIDS, we conclude that it reflects HCO3- uptake into the cells via a DIDS sensitive Cl-/HCO3- exchanger which alkalinizes the cells and stimulates the basolateral K+ conductance. Our results confirm, revise and extend the results of previous, less direct, investigations of gastric cell ion transport.

Twenty strains of Streptococcus thermophilus were classified into five different types characterized by ribotyping after DNA digestion with Hin dIII and hybridization with cDNA from 16S–23S rDNA of Escherichia coli . Ribotyping after digestion with Hae III and Xho l 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 subtaca are to be recognized within this species.

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.

Randomly amplified polymorphic DNA analysis using primer 239 (5′ CTGAAGCGGA 3′) was performed to characterize Leuconostoc sp. strains. All the strains of Leuconostoc mesenteroides subsp. mesenteroides (with the exception of two strains), two strains formerly identified as L. gelidum , and one strain of Leuconostoc showed a common band at about 1.1 kb. This DNA fragment was cloned and sequenced in order to verify its suitability for identifying L. mesenteroides subsp. mesenteroides strains.

In this paper we analyze the crystallization kinetics under steady shear flow conditions of different samples obtained by blending two isotactic poly(1-butene)s with different average molecular weights. It is observed that the addition of a small amount of high molecular weight (MW) polymer (

Lactic acid bacteria occurring in Provolone del Monaco, an artisanal pasta filata cheese produced in Campania (Italy) from raw cows’ milk and without starter addition, were investigated by a combination of conventional and molecular approaches. The microbial community was monitored during a cheese-making process giving rise to a premium quality product. Streptococcus thermophilus and Streptococcus macedonicus prevailed during cheese manufacture and survived along nine months of ripening, together with enterococci and lactobacilli of the casei group, especially Lactobacillus rhamnosus. Phenotypic and genetic identification of 308 isolates largely reflected the results obtained by 16S rDNA sequencing analysis by polymerase chain reaction-denaturant gradient gel electrophoresis, with the significant exception of Lactobacillus fermentum and four Lactobacillus delbrueckii subspecies that were not detected by cultural methods. Each different analytical approach employed provided useful information. Their combination proved to be suitable to effectively describe the ecosystem of Provolone del Monaco cheese.

Although the use of starter cultures in winemaking has improved the reproducibility and predictability of wine quality, the main drawback to this practice is the lack of the typical traits of wines produced by spontaneous fermentation. In this study, we identified for the first time the yeast population occurring during spontaneous fermentation of the Catalanesca white grape, a variety from Campania (Italy). Yeasts were identified using molecular tools: PCR-DGGE and partial sequence analysis of the 26S rRNA gene from isolates. Eighteen different species belonging to 11 different genera were identified. Hanseniaspora spp., Issatchenkia spp. and Candida spp. were the dominant yeasts during the early stages of fermentation, while the middle and end phases were dominated by Saccharomyces cerevisiae. Four species of Issatchenkia spp., rarely isolated from wine fermentation, were found in this study accounting for the 33.5% of the total isolates. The RAPD-PCR screening of the isolates followed by partial rRNA gene sequencing proved to be a very effective approach to first differentiate the isolates and then identify yeast species involved in a wine making procedure. The results show very high yeast diversity in this natural wine fermentation and also highlight the possibility of considering interesting autochthonous strains for starter selection.

The effect of ammoniac deoxidizing agent (Urea) on the reduction of NO x produced in the Diesel engine was investigated numerically. Urea desolved in water was directly injected into the engine cylinder during the expansion stroke. The NOx deoxidizing process was described using a simplified chemical kinetic model coupled with the comprehensive kinetics of Diesel oil surrogate combustion. If the technology of DWI (Direct Water Injection) with the later injection timing is supposed to be used, the deoxidizing reactants could be delivered in a controlled amount directly into the flame plume zones, where NOx are forming. Numerical simulations for the Isotta Fraschini DI Diesel engine are carried out using the KIVA-3V code, modified to account for the "co-fuel" injection and reaction with combustion products. The results showed that the amount of NOx could be substantially reduced up to 80% with the injection timing and the fraction of Urea in the solution optimized. Copyright © 2007 SAE International.

Microbial diversity of the raw milk for the production of Fior di Latte di Agerola and its changes during cheesemaking were studied. Viable counts showed that at the end of curd ripening, loads of lactic acid bacteria, both mesophilic and thermophilic rods and cocci, higher than those commonly evidenced in similar cheeses produced by using natural or commercial starters, were detected. Identification of 272 isolates, supported by molecular diagnostic aids, evidenced representative cultures of a high number of bacterial taxa of interest as participating in the process, although most of the isolates belonged to Lactococcus lactis and Lactobacillus helveticus species. RAPD-PCR and REA-PFGE biotyping were performed for the isolates of the above species and it was shown that most of the strains isolated from the raw milk occurred during the whole cheesemaking process, and an active role of these strains in the fermentation was supposed. The results offer further proof of the importance of the raw milk as source of technologically interesting strains of lactic acid bacteria capable of driving the fermentation of traditional cheeses.

Using rheological techniques, we investigate the evolution of the microstructure evolution during the early stages of crystallization of poly(1-Butene). In performing the measurements, use is made of an innovative experimental protocol, called “inverse quenching”, which allows stopping the crystallization process and producing a stable biphasic (crystalline/amorphous) system. In this way, very low frequency measurements at fixed degrees of crystallization are made possible. We find that crystallization, evidenced as a Liquid-to-Solid Transition (LST) under isothermal conditions, with characteristics of critical gel behavior, takes place at surprisingly low degrees of crystallinity (below 1.5%). The critical gel properties, which are found to depend on both crystallization temperature and molecular weight, can be reduced to a single master curve when the gel strength is plotted as a function of the relaxation exponent.. More importantly, the LST it is preceded by the development of a long, but finite relaxation process. This latter process, although not fully understood, brings analogies to the slow dynamics observed in hybrid colloid-polymer systems (block copolymer micelles or multiarm star polymers) as well as the recently suggested presence of dormant nuclei. It is clear, however, that the connectivity among crystallites, apparently via the amorphous segments, plays a key role in this new process

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.

D . E R C O L I N I , G . M A U R I E L L O , G . B L A I O T T A , G . M O S C H E T T I A N D S . C O P P O L A . 2003. 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� 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.

Flow Induced Crystallization (FIC) is the common term to indicate the acceleration in polymer crystallization kinetics due to the action of flow. When modeling FIC, two major challenges are encountered. On the one hand, the model must be able to produce quantitative reliable results, while correctly describing the coupling between the intrinsic (quiescent) crystallization kinetics and the rheological response of the polymer. On the other hand, the model must be able to describe the complex kinematics taking place in real industrial processes. In this paper, we present the predictions of a recently proposed model for FIC in the case of a mixed flow, where both shear and extensional components are present at the same time. In particular, the effects of the overall flow intensity and of relative weight between shear and extension on the enhancement in nucleation rate are presented and discussed. Some guidelines for future development are also proposed.

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 104 CFU ml−1 by amplification of nuc and seh genes. 105 and 107 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.

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.

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.

The modified Chalmers medium appeared as quite suitable to counting a mixing 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. The modified Chalmers medium has proved to be very satisfactory for differential counts of the main species of lactic acid bacteria occurring in starter for dough leavening process

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. Conclusions: 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.

Separation of amplified V3 region from 16S rDNA by denaturing gradient gel electrophoresis (DGGE) was tested as a tool for differentiation of lactic acid bacteria commonly isolated from food. Variable V3 regions of 21 reference strains and 34 wild strains referred to species belonging to the genera Pediococcus, Enterococcus, Lactococcus, Lactobacillus, Leuconostoc, Weissella, and Streptococcus were analyzed. DGGE profiles obtained were species-specific for most of the cultures tested. Moreover, it was possible to group the remaining LAB reference strains according to the migration of their 16S V3 region in the denaturing gel. The results are discussed with reference to their potential in the analysis of LAB communities in food, besides shedding light on taxonomic aspects.

The problem of Flow Induced Crystallization (FIC) of polymer melts is addressed via a micro-rheological approach. In particular, the Doi-Edwards model with the so-called Independent Alignment Approximation (DE-IAA) is used to calculate the flow-induced change of free energy. Subsequently, the crystallization induction time, i.e., the nucleation characteristic time, is calculated in isothermal steady shear and uniaxial elongational flows. Asymptotic, analytical expressions for the induction time are also derived in the limit of low and high Deborah number (the product of the deformation rate and the polymer relaxation time). The DE-IAA model is found to give more realistic predictions than those of simpler, dumbbell-like models already proposed in the literature. When compared to existing FIC experimental data in shear flow, good quantitative agreement is found with the polymer relaxation time as the only adjustable parameter of the model.