Setup of a rapid method to distinguish among dead, alive, and viable but not cultivable cells of Pseudomonas spp. in mozzarella cheese.

Pseudomonas spp. is the main psychrotrophic genus involved in the spoilage of raw milk and more in general of dairy products, such as mozzarella cheese. The members of this bacterial species are able to produce heat-resistant proteolytic enzymes, determining the casein hydrolysis, and as a consequence, a reduction of the shelf life and sensory quality of the products. Therefore, the spoilage activity could be attributed not only to viable, but also to viable but noncultivable (VBNC) cells. For this reason, the setup of a non-culture-based method is useful for a rapid detection of cells that are still alive, but no longer cultivable, such as VBNC cells. Here we propose a method based on DNA or RNA content (or both) to reveal the presence of dead, alive, and VBNC cells belonging to the genus Pseudomonas. The obtained results clearly indicate the limits of the classical plating count overcome by molecular detection of Pseudomonas spp. through DNA and RNA analysis, enabling us to establish the presence of different states of the cells.

Effect of storage temperature on the microbial composition of ready-to-use vegetables.

Four different salad preparations were investigated from microbiological point of view: two were packaged in air and two under Modified Atmosphere. The samples were stored at 4 and 10 °C, and analysed at established times. Total bacterial count (TBC) was taken as the most relevant index to define their hygiene and quality at both temperatures. Lactic acid bacteria, yeasts and moulds were found only occasionally. In general, the most important factor was the packaging technique: TBC was lower when the product is packed under modified conditions. The packaging technique also influences the microbial population: Gram-negative aerobic rods are dominant in air-packaged products, whilst the presence of Enterobacteriaceae becomes important in salads packaged under Modified Atmosphere. Pseudomonas fluorescens, with all its biovars, was the most frequently found species amongst the aerobic isolates, whilst for the Enterobacteriaceae strains, there was no dominant species.

Shelf life of case-ready beef steaks (Semitendinosus muscle) stored in oxygen-depleted master bag system with oxygen scavengers and CO2/N2 modified atmosphere packaging.

This study aims to evaluate the stability of beef from Semitendinosus muscle packaged in oxygen permeable wrapped-tray units and stored in a master bag system, with and without oxygen scavengers. Changes in the atmosphere composition, microbiological indexes, myoglobin forms and color parameters were monitored during the storage in master bag, blooming and display life. The presence of scavengers reduced rapidly the oxygen concentration and maintained it at values not detectable instrumentally. Within few days of storage in master bags, the resolution of the transient discoloration was completed and the meat quality was maintained over the anoxic storage. After the removal from master bags meat bloomed completely reaching OxyMb level and Chroma values higher than those on fresh meat at t(0). During 48 h of display life at 4 °C, quality attributes had a decay slower than samples stored traditionally in air. Without scavengers the oxygen caused the irreversible discoloration within 7 days, due to the formation of metmyoglobin on the surface.

Phenotypic and genotypic characterization of lactic acid bacteria isolated from Artisanal Italian goat cheese.

The lactic acid bacteria community in traditional goat cheese produced in three dairies in Valsesia (Piemonte, Italy) was studied at different steps of the manufacturing process. These cheeses were produced from raw milk without starter bacteria, and no protocol was followed during the manufacturing process. Three hundred thirty-two isolates were characterized and grouped by results of both morphophysiological tests and random amplification of polymorphic DNA plus PCR analysis. Bacteria were identified by partial sequencing of the 16S rRNA gene. Lactococci were the dominant lactic acid bacteria in raw milk. Their initial numbers ranged from 5 to 7 log CFU ml(-1). Their levels increased during manufacturing and decreased during ripening. The growth trend for enterococci was comparable to that of lactococci, although enterococci counts were lower. Lactococcus lactis subsp. cremoris, Lactococcus garviae, and Enterococcus faecalis were the most frequently isolated species during goat cheese manufacturing, whereas the highest numbers of Enterococcus (E. faecium, E. durans, E. gilvus, and E. casseliflavus) were isolated with the greatest frequency from ripened cheese samples. Occasionally, Leuconostoc mesenteroides, Leuconostoc lactis, and Lactobacillus paraplantarum also were isolated.

Evaluation and predictive modeling of shelf life of minced beef stored in high-oxygen modified atmosphere packaging at different temperatures.

The aims were: (1) to follow the freshness decay of minced beef stored in high-oxygen modified atmosphere packaging at different temperatures (4.3, 8.1 and 15.5 degrees C) by applying traditional methods (microbiological counts, color evaluation, thiobarbituric acid assay TBA, headspace gas composition) and e-nose; (2) to model the decay kinetics to obtain information about the maximum shelf life as function of storage conditions. The minced beef, packaged in modified atmosphere was supplied by a manufacturer at the beginning of its commercial life. The study demonstrated the ability of the traditional methods to describe the kinetics of freshness decay. The modeling of the experimental data and the comparison with microbiological or chemical thresholds allowed the setting, for each index, of a stability time above which the meat was no longer acceptable. The quality decay of meat was also evaluated by the headspace fingerprint of the same set of samples by means of a commercial e-nose. A clear discrimination between "fresh" and "old" samples was obtained using PCA and CA, determining at each temperature a specific range of stability time. The mean value of the stability times calculated for each index was 9 days at 4.3 degrees C (recommended storage temperature), 3-4 days at 8.1 degrees C (usual temperature in household refrigerators) and 2 days at 15.5 degrees C (abuse temperature). Resolution of the stability times allowed calculation of mean Q(10) values, i.e. the increase in rate for a 10 degrees C increase in temperature. The results show that the Q(10) values from the traditional methods (3.6-4.0 range) overlapped with those estimated with e-nose and color indexes (3.4 and 3.9, respectively).

Microbiological quality and shelf life modeling of ready-to-eat cicorino.

Growth of the microbial population resident in ready-to-use fresh cut cicorino, a variety of Chichorium intybus, was determined in the various preparation steps with the aim of defining the hazard critical control points. The investigation concerned cut cicorino from two producers. During the process a 1- to 1.5-fold increase of microbial counts was observed, and the retail product showed values of 10(5) to 10(6) CFU/g. The shelf life of the product was kinetically modeled in order to check the effects of storage temperature and assess the microbial indexes most relevant for hygiene and quality of the distributed product. A modified Gompertz function described the kinetics of microbial growth and allowed definition of a stability time and its dependence on temperature. Stability times were of 0.3, 3.7, and 4.7 days at storage temperatures of 20, 10, and 5 degrees C, respectively. Q10 (the fold decrease of stability time for an increase of 10 degrees C) was 3.85. The results from this study may be used to predict the effects of temperatures experienced in the distribution chain on bacterial levels in cicorino.

Production of cyclopiazonic acid by molds isolated from Taleggio cheese.

Twenty-seven strains of Penicillium were isolated from the rind of Taleggio, a typical Italian cheese, so that we could test their capacity to produce cyclopiazonic acid (CPA); all strains produced CPA. The production was strongly influenced by the strain variety and growth conditions. Strains incubated at 25 degrees C for 7 days always produced CPA in mannitol broth, with concentrations ranging from 0.02 to 1 microg/ml, whereas only 33% of strains grown in yeast-extract broth produced CPA, with a maximum value of 0.1 microg/ml. In milk, maximum production (1.6 microg/ml) was observed after 14 days of incubation at 25 degrees C. In order to evaluate the presence of the toxin and its capacity for migrating into the cheeses, the rind, the cheese near the rind, and the cores from six Taleggio cheeses were analyzed. CPA was present in five cheeses, with a maximum concentration of 0.25 mg/kg in one rind, and in one cheese, the toxin migrated to the core. A positive correlation between CPA production and surface mold was found.

Repair of periodontal furcation defects in beagle dogs following reconstructive surgery including root surface demineralization with tetracycline hydrochloride and topical fibronectin application.

This study examines the effects of root surface demineralization and topical fibronectin as adjuncts to reconstructive periodontal surgery. In 14 beagle dogs, horizontal periodontal defects were surgically induced around the mandibular premolars followed by a 6-week period without plaque control. Reconstructive surgery of the defects was subsequently carried out. The root surfaces were debrided and superficially demineralized with citric acid or tetracycline hydrochloride, with or without subsequent application of fibronectin. Mucoperiosteal flaps were raised to cover most of the crowns and sutured. The animals were sacrificed 12 weeks after surgery and block sections of the teeth and surrounding tissues were processed for histology. Analysis included incidence of furcation defects presenting with an epithelial lining, quantification of connective tissue repair relative to the furcation circumference, and regeneration of alveolar bone relative to the furcation defect height. The incidence of root resorption and ankylosis was also analyzed. Within the limitations of this study it was concluded that: (1) citric acid conditioning of the root surface frequently resulted in complete connective tissue repair of the furcation defect; (2) root resorption and ankylosis were prevalent features of the healing response; (3) citric acid and tetracycline treatment had similar potential to induce connective tissue repair and resulted in corresponding incidences of root resorption and ankylosis; (4) application of fibronectin to demineralized root surfaces did not enhance the amount of connective tissue repair and did not alter the pattern of root resorption and ankylosis.

A biochemical approach to periodontal regeneration. AFSCM: assays for specific cell migration.

Recent investigations on regeneration of the periodontium have attempted to define factors involved in the formation of a new connective tissue attachment. One essential biological event involved in tissue regeneration is directed cell migration (chemotaxis). Extracellular matrix proteins have been shown to influence chemotaxis, cell proliferation and differentiation. Recently, the extracellular matrix proteins, fibronectin (FN) and laminin (LM), and the polypeptide, endothelial cell growth factor (ECGF), have been shown to stimulate a variety of biological processes. Current assay systems which attempt to define cell migration are the Boyden chamber assay and a random cell migration assay. Neither assay system adequately defines in vivo cell migration. Here we present a new in vitro assay system that tests the capacity of several biological response modifiers applied on dentin to stimulate a chemotactic and proliferative response from various cell types. The assay system consists of two types of assays. Assay I measures the chemotactic activity of test substances bound to dentin. In this assay cells must actively move through a filter (Nuclepore) towards a factor bound to dentin. Assay II examines the ability of dentin-bound biological response modifiers to stimulate directed movement and proliferation of cells on dentin surfaces. We report that periodontal ligament (PDL) cells migrate towards FN and ECGF; that PDL cell migration is enhanced when dentin is preconditioned with tetracycline HCl; that PDL cells have an increased proliferative response when dentin is conditioned with both FN and ECGF; that gingival epithelial cells have increased migratory and proliferative responses when LM is used to condition dentin; and that there is a reciprocal utilization of biological response modifiers by gingival epithelial cells and PDL cells.