Assessment of safety of enterococci isolated throughout traditional Terrincho cheesemaking: virulence factors and antibiotic susceptibility

Enterococci account for an important fraction of the adventitious microflora of traditional cheeses manufactured in Mediterranean countries from small ruminants' raw milk and play an important role in the development of suitable organoleptic characteristics of the final product. It has been suggested that animals used for food or animals that supply edible products are a reservoir of antibiotic-resistant enterococci. The main purpose of this research effort was thus to identify, to the species level, a total of 73 enterococci with high tolerance to acidic pH and bile salts (as prevailing environmental conditions in the first portion of the gastrointestinal tract), which were previously isolated from the milk feedstock to the final product of Terrincho cheesemaking, and to determine their profiles of antibiotic susceptibility, coupled with the occurrence of specific virulence factors (especially in those that might eventually be claimed to exhibit suitable probiotic and technological performances). Isolates, identified by both API 20 STREP and PCR methods, were found to belong to the following Enterococcus species: E. casseliflavus, E. durans, E. faecalis, E. faecium, and E. gallinarum. Susceptibility of those isolates was observed to most antibiotics tested, whereas none harbored aminoglycoside resistance genes. PCR screenings for cytolysin genes (cylL(L), cylL(s), cylM, cylB, and cylA), surface adhesin genes (efaA(fs), efaA(fm), and esp), the aggregation protein gene (agg), and the extracellular metalloendopeptidase gene (gelE) were performed. All isolates proved negative for cylL(L), cylM, cylB, and agg genes. Both E. faecalis strains were positive for the cell wall-associated protein Esp and the cell wall adhesin efaA(fs), whereas the cell wall adhesin efaA(fm) was detected in 11 of the 12 E. faecium strains. Only one strain possessed the cylL(s) determinant, and another possessed the cylA gene. Incidence of virulence determinants was thus very low; hence, the enterococcal adventitious microflora tested is essentially safe.     

Isolation of an acetyl-CoA synthetase gene (ZbACS2) from Zygosaccharomyces bailii

A gene homologous to Saccharomyces cerevisiae ACS genes, coding for acetyl-CoA synthetase, has been cloned from the yeast Zygosaccharomyces bailii ISA 1307, by using reverse genetic approaches. A probe obtained by PCR amplification from Z. bailii DNA, using primers derived from two conserved regions of yeast ACS proteins, RIGAIHSVVF (ScAcs1p; 210-219) and RVDDVVNVSG (ScAcs1p; 574-583), was used for screening a Z. bailii genomic library. Nine clones with partially overlapping inserts were isolated. The sequenced DNA fragment contains a complete ORF of 2027 bp (ZbACS2) and the deduced polypeptide shares significant homologies with the products of ACS2 genes from S. cerevisiae and Kluyveromyces lactis (81% and 82% identity and 84% and 89% similarity, respectively). Phylogenetic analysis shows that the sequence of Zbacs2 is more closely related to the sequences from Acs2 than to those from Acs1 proteins. Moreover, this analysis revealed that the gene duplication producing Acs1 and Acs2 proteins has occurred in the common ancestor of S. cerevisiae, K. lactis, Candida albicans, C. glabrata and Debaryomyces hansenii lineages. Additionally, the cloned gene allowed growth of S. cerevisiae Scacs2 null mutant, in medium containing glucose as the only carbon and energy source, indicating that it encodes a functional acetyl-CoA synthetase. Also, S. cerevisiae cells expressing ZbACS2 have a shorter lag time, in medium containing glucose (2%, w/v) plus acetic acid (0.1-0.35%, v/v). No differences in cell response to acetic acid stress were detected both by specific growth and death rates. The mode of regulation of ZbACS2 appears to be different from ScACS2 and KlACS2, being subject to repression by a glucose pulse in acetic acid-grown cells.     

Effects of freezing and frozen storage on the physicochemical and sensory characteristics of four types of goat's cheese

Summary Physicochemical and organoleptic properties were studied in two batches of four different types of goat's milk cheese: fresh, washed curd, soft with surface flora, and Majorero, frozen for four months prior to ripening and/or chilled storage. Frozen storage did not produce significant changes in the rheological or sensory characteristics of the cheeses, except in the case of fresh cheese, the textural characteristics of which were adversely affected. The level of proteolysis, estimated from the amino-acid nitrogen, was higher in the pre-frozen washed curd, soft with surface flora, and Majorero cheeses. The level of lipolysis was comparable, except in the soft cheese with surface flora, which presented higher levels of free fatty acids when stored frozen before ripening.

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Einfluß des Gefrierens und der Gefrierlagerung auf die physikalisch-chemischen und organoleptischen Eigenschaften von vier Ziegenkäsen

Zusammenfassung Aus Ziegenmilch wurden jeweils zwei Chargen von vier spanischen Käsesorten hergestellt: Frischkäse, gewaschene Käsemasse, Weichkäse mit Oberflächenflora und Majorero -Käse. Von jeder Charge wurde die Hälfte gefroren gelagert und erst nach vier Monaten auf die übliche Weise gereift und/oder gekühlt aufbewahrt. Die physikalisch-chemischen und organoleptischen Eigenschaften wurden danach untersucht. Im Vergleich mit Käsen, die nicht gefroren gewesen waren, ergaben sich keine wesentlichen Unterschiede in den rheologischen und sensorischen Eigenschaften, mit Ausnahme bei den Frischkäsen, bei dem sich die Textur durch die Aufbewahrung in gefrorenem Zustand verschlechtert hatte. Der Gehalt an freien Aminosäuren, ein Maß für die Proteolyse, war höher in der gewaschenen Käsemasse, in dem Weichkäse mit Oberflächenflora und in dem Majorero-Käse, die vier Monate gefroren gewesen waren. Durch die Gefrierlagerung hatte sich der Fettabbau nicht verändert, mit Ausnahme bei dem Käse mit Oberflächenflora, bei dem der Gehalt an freien Fettsäuren erhöht war im Vergleich mit dem Käse, der nicht vier Monate gefriergelagert worden war.

     

Processing and analytical characterisation of pulp-enriched cloudy apple juices

Pulp-enriched cloudy apple juices were manufactured from two apple varieties by blending of apple puree with natural cloudy apple juice in order to enrich bioactive secondary plant substances. Finishing of the puree with a 0.6-mm mesh screen and final product homogenisation revealed as the optimum processing technology for the novel 100% fruit beverage. The presence of large particles originating from the puree prevented long-term cloud stability, but due to the proportion of cloud-stable juice a complete phase separation did not occur. Optimal drinkability was achieved at a viscosity of 11.5 mPa s. Polyphenol compositions and concentrations were determined by means of RP-HPLC/PDA. The results ranged from 109 to 610 mg/l. All samples showed the typical polyphenol pattern of apples with dominating hydroxycinnamic acids followed by flavanols and flavonols. The puree addition to the cloudy apple juices increased the polyphenol concentrations by average 100%. Relatively, the highest increase could be observed for dimeric procyanidins. Different technological variants did not affect significantly the polyphenol concentrations. The dietary fibre contents of the pulp-enriched cloudy apple juices ranged from 5.8 to 9.4 g/l.     

Syrah grape skin valorisation using ultrasound-assisted extraction: Phenolic compounds recovery,antioxidant capacity and phenolic profile

The influence of ultrasound power (1000–3000 W/L), citric acid concentration (0–3%) and solid:liquid ratio (1:5–1:15) on the phenolic compounds recovery and antioxidant capacity of Syrah grape skin extracts were evaluated. Total phenolic compounds varied from 6485 to 11732 mg gallic acid/100 g and monomeric anthocyanin content from 453 to 685 mg malvidin-3-glucoside/100 g. The antioxidant capacity measured by ORAC and ABTS methods ranged from 230 to 516 μmol Trolox/g and from 442 to 939 μmol Trolox/g, respectively. The most suitable conditions chosen for extraction, within the studied ranges, were 3000 W/L of power, 2.5% citric acid and solid:liquid ratio of 1:15. The extraction yield was satisfactory, with a recovery of 59% of the quantified phenolic compounds, with only 3 min of processing. Ultrasound was considered a suitable method as compared to the conventional extraction, improving the extraction of phenolic acids and facilitating their release.     

Rheological properties and baking quality of commercial durum wheat meals used to make flat crispy bread

Four commercial semolinas and four low-grade semolinas (LGS) samples were used in this study to make flat crispy bread (Carasau). The meals, doughs, and bread were compared with respect to a set of physical, chemical, and textural characteristics, and the bread evaluated by a trained sensory panel. LGSs had a higher extraction rate (higher ash content), finer granularity, and a higher damaged starch content than semolinas. The study revealed the key role of dry gluten content in determining hydration capacity of the dough, whereas damaged starch affected water absorption only in semolina samples. Gluten index and the ratio of gluten tenacity to extensibility (alveograph P/L) were significantly correlated with the weight of the dough discs following sheeting and molding. The panelists gave the highest quality score to the bread with the highest values of crackling and hardness. Textural and sensory attributes were both correlated with one another, and with gluten strength and tenacity, but not with the gluten content of the meal, which only affected final moisture content of the bread. Meal samples with a more tenacious and elastic gluten were more suited to produce flat crispy breads. The yellowness of durum wheat bread could not be predicted by measuring the color of the meal with a reflectance method.     

Changes in selected biochemical and sensory parameters as affected by the superficial inoculation of Penicillium camemberti on dry fermented sausages

An atoxigenic strain of Penicillium camemberti was superficially inoculated on fermented sausages in an attempt to improve their sensory properties. The growth of this mould on the surface of the sausages resulted in an intense proteolysis and lipolysis, which caused an increase in the concentration of free amino acids, free fatty acids (FFA) and volatile compounds. Many of these were derived from amino acid catabolism and were responsible for the "ripened flavour", i.e. branched aldehydes and the corresponding alcohols, acids and esters. The development of the fungal mycelia on the surface of the sausages also protected lipids from oxidation, resulting in both lower 2-thiobarbituric acid (TBARS) values and lipid oxidation-derived compounds, such as aliphatic aldehydes and alcohols. The sensory analysis of superficially inoculated sausages showed clear improvements in odour and flavour and, as a consequence, in the overall quality of the sausages. Therefore, this strain is proposed as a potential starter culture for dry fermented sausage production.     

Use of carbon dioxide enriched atmospheres in the refrigerated storage (2 °C) of salmon (Salmo salar ) steaks

 Selected microbial counts (total viable microbiota, Brochothrix thermosphacta, lactic acid bacteria and Enterobacteriaceae), biochemical parameters [pH, total volatile nitrogen, nucleotide breakdown products, non-volatile amines, d(–) and l(+)-lactic acids and short-chain fatty acids] and sensory attributes (colour and odour) of refrigerated (2  °C) salmon (Salmo salar) steaks stored under CO₂-enriched [CO₂/air (20/80, v/v), CO₂/air (40/60, v/v)] and air atmospheres were determined. When compared with air, sensory results showed shelf-life extension of 6 days and 15 days for 20% and 40% CO₂-enriched atmospheres, respectively. Microbial and biochemical results also revealed that the 40% CO₂-enriched atmosphere was the most effective packaging type for refrigerated salmon. Received: 22 April 1999     

Volatile compound generation in dry fermented sausages by the surface inoculation of selected mould species

The effect of the inoculation of dry fermented sausage surface with an atoxigenic, proteolytic and lipolytic strain of Mucor racemosus, Penicillium aurantiogriseum and Penicillium camemberti on the volatile composition was studied. The analysis of the headspace volatile compounds using gas chromatography/mass spectrometry enabled the identification of 55 volatiles. The study showed that every mould species produced a different volatile profile which was also different from that of the control sausages. Compounds derived from amino acid catabolism, i.e. branched aldehydes and the corresponding alcohols, were produced in higher amounts in sausages inoculated with Penicillium spp. On the other hand, volatiles coming from the microbial esterification were related to sausages inoculated with M. racemosus. The development of the fungal mycelia on the sausage surface protected lipids from oxidation, thus giving rise to fewer lipid oxidation products in the inoculated sausages.     

Behavior of Yersinia enterocolitica strains inoculated in model cheese treated with high hydrostatic pressure

The effects of high hydrostatic pressure treatment and the ability for survival, repair, and growth of three human pathogenic serotypes (O:1, O:3, O:8) of Yersinia enterocolitica were investigated in washed-curd model cheese made with pasteurized bovine milk. Samples were treated at 300, 400, and 500 MPa for 10 min at 20 degrees C and analyzed at 0, 1, 7, and 15 days to assess the viability of the Yersinia population. A long-term study (up to 60 days of ripening after high hydrostatic pressure treatment) was also undertaken. Treatments at 400 and 500 MPa caused maximum lethality, and only the treatment at 300 MPa showed significant differences (P < 0.05) between serotypes; the most baroresistant was O:3. Ability to repair and grow was not observed after 15 days of storage at 8 degrees C. Yersinia counts in untreated cheese samples also decreased below the detection limit at day 45 in the long-term study. These results suggest that the cheese environment did not allow recovery of injured cells or growth. A primary contributing factor to this effect seemed to be the low pH resulting from the production of lactic acid during cheese ripening.