Marilena Marino

Impedance measurements to study the antimicrobial activity of essential oils from Lamiaceae and Compositae

A wide range of essential oils from sage, mint, hyssop, camomile and oregano were tested for their inhibitory effects against nine strains of gram-negative bacteria and six strains of gram-positive bacteria. Three principles were used in describing the antimicrobial effects of the essential oils: the overall antimicrobial activity determined by use of an impedometric method, the bactericidal effect determined as colony forming units after exposure to the essential oils, and the number of apparent dead cells determined after further enrichment. The data obtained indicate that while the essential oils of sage, mint, hyssop and camomile had generally a bacteriostatic activity, the essential oil from oregano appeared to be bactericidal at concentrations above 400 ppm, probably because of high contents in phenolic compounds. For the other essential oils, the chemical analysis was unable to explain the antimicrobial effect. The bacteriostatic activity was more marked against gram-positive bacteria; in contrast, the bactericidal activity was greatest against gram-negative bacteria. The most sensitive strain was Escherichia coli O157:H7 and, of the gram-positive species even at the lowest oil concentrations, Listeria innocua was the most sensitive. The data obtained from the study of the bactericidal effect of oregano essential oil indicated that the major part of the species was irreversibly inactivated, i.e. they could not be revived by enrichment.

Antimicrobial activity of the essential oils of Thymus vulgaris L. measured using a bioimpedometric method

The essential oils obtained from Thymus vulgaris L. harvested at four ontogenetic stages were evaluated for their biological activity and chemical composition. The thyme essential oils were tested for their inhibitory effects against nine strains of gram-negative bacteria and six strains of gram-positive bacteria. The bioimpedance method was chosen for studying the antibacterial activity of the essential oils and the parameter chosen for defining and quantifying the antibacterial activity of the essential oils was the detection time. The plate counting technique was used to study the inhibitory effect by direct contact. All the thyme essential oils examined had a significant bacteriostatic activity against the microorganisms tested. This activity was more marked against the gram-positive bacteria. The oil from thyme in full flower was the most effective at stopping the growth of the microbial species examined. The oils tested were also shown to have good antibacterial activity by direct contact, which appeared to be more marked against the gram-negative bacteria. Only a few of the species were capable of recovering at least 50% of their metabolic function after contact with the inhibitor, while most of the strains were shown to have been inactivated almost completely. Escherichia coli O157:H7 was the most sensitive species, given that after contact with even the lowest concentration of oil cells could not be recovered.

The capacity of Enterobacteriaceae species to produce biogenic amines in cheese

The amino acid decarboxylating activity and production of biogenic amines by 104 cheese‐associated Enterobacteriaceae species (58 Enterobacter, 18 Serratia, eight Escherichia, seven Hafnia, six Arizona, four Citrobacter and three Klebsiella) were investigated. All strains could decarboxylate at least two amino acids in Möllers broth and in Nivens medium, and the decarboxylase activity was strain specific. In a laboratory medium containing all free amino acids, all strains could produce more than 100 ppm cadaverine, putrescine was produced by 96% of strains. Tyramine and histamine were produced in the lowest concentrations. A positive correlation existed between cadaverine concentration and Enterobacteriaceae counts in cheese, that may have caused the increase in decarboxylase content. This study suggests that it is possible to limit the presence of cadaverine in cheese, thereby controlling the Enterobacteriaceae counts, a sign of contamination during cheese making and/or storage.

Microbiological characterization of artisanal Montasio cheese: analysis of its indigenous lactic acid bacteria

The aim of this study was to investigate the dynamics of the microflora during Montasio cheese ripening, with specific reference to some characteristics of biotechnological interest. Nine batches of Montasio cheese produced in different plants were analyzed. Streptococcus thermophilus was the predominant species throughout the whole ripening period of Montasio cheese. Enterococci were also frequently present. This microbial group resulted probably from milk, and its proportion decreased rapidly during ripening. The most acidifying microbial species was S. thermophilus, while the most proteolytic strains belonged to the genera Enterococcus. A high degree of phenotypic diversity occurred within the microbial species.

Comparison of culture-dependent and -independent methods for bacterial community monitoring during Montasio cheese manufacturing

The microbial community in milk is of great importance in the manufacture of traditional cheeses produced using raw milk and natural cultures. During milk curdling and cheese ripening, complex interactions occur in the microbial community, and accurate identification of the microorganisms involved provides essential information for understanding their role in these processes and in flavor production. Recent improvements in molecular biological methods have led to their application to food matrices, and thereby opened new perspectives for the study of microbial communities in fermented foods. In this study, a description of microbial community composition during the manufacture and ripening of Montasio cheese was provided. A combined approach using culture-dependent and -independent methods was applied. Culture-dependent identification was compared with 16S clone libraries sequencing data obtained from both DNA and reverse-transcribed RNA (cDNA) amplification and real-time quantitative PCR (qPCR) assays developed to detect and quantify specific bacterial species/genera (Streptococcus thermophilus, Lactobacillus casei, Pediococcus pentosaceus, Enterococcus spp., Pseudomonas spp.). S. thermophilus was the predominant LAB species throughout the entire ripening period of Montasio cheese. The culture-independent method demonstrates the relevant presence of Pseudomonas spp. and Lactococcus piscium at the beginning of ripening. The culture-dependent approach and the two culture-independent approaches produced complementary information, together generating a general view of cheese microbial ecology.

Safety‐related properties of staphylococci isolated from food and food environments

Aims:  To test some safety‐related properties within 321 staphylococci strains isolated from food and food environments.

Evaluation of amino acid-decarboxylative microbiota throughout the ripening of an Italian PDO cheese produced using different manufacturing practices

Aim:  To investigate the presence of biogenic amines (BAs) in Montasio cheese produced by using different cheese manufacturing practices.

Influence of hydroponic and soil cultivation on quality and shelf life of ready‐to‐eat lamb's lettuce (Valerianella locusta L. Laterr)

BACKGROUND Nowadays, there is an increasing interest in the hydroponic floating system to cultivate leafy vegetables for ready-to-eat salads. It is reasonable that different growing systems could affect the quality and shelf life of these salads. RESULTS The quality and shelf life of ready-to-eat lambs lettuce grown in protected environment in soil plot or in soil-less system over hydroponic solution with or without the addition of 30 µmol L⁻¹ silicon were evaluated. Minimum effects were observed on colour, firmness and microbial counts. Hydroponic cultivation largely affected plant tissue hydration, leading to weight loss and structural modifications during refrigerated storage. The shelf life of lambs lettuce was limited by the development of visually detectable unpleasant sensory properties. Shelf life, calculated by survival analysis of consumer acceptability data, resulted about 7 days for soil-cultivated salad and 2 days for the hydroponically grown ones. The addition of silicon to the hydroponic solution resulted in an interesting strategy to increase plant tissue yield and reduce nitrate accumulation. CONCLUSIONS Although hydroponic cultivation may have critical consequences on product quality and shelf life, these disadvantages could be largely counterbalance by increased yield and a reduction of nitrate accumulation when cultivation is performed on nutritive solutions with supplemental addition of silicon.

Identification of the Enterobacteriaceae in Montasio cheese and assessment of their amino acid decarboxylase activity.

The aim of the study was to identify the species of Enterobacteriaceae present in Montasio cheese and to assess their potential to produce biogenic amines. Plate count methods and an Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) approach, combined with 16S rDNA sequencing, were used to investigate the Enterobacteriaceae community present during the cheesemaking and ripening of 6 batches of Montasio cheese. Additionally, the potential decarboxylation abilities of selected bacterial isolates were qualitatively and quantitatively assessed against tyrosine, histidine, ornithine and lysine. The most predominant species detected during cheese manufacturing and ripening were Enterobacter cloacae, Escherichia coli and Hafnia alvei. The non-limiting physico-chemical conditions (pH, NaCl% and a(w)) during ripening were probably the cause of the presence of detectable levels of Enterobacteriaceae up to 120 d of ripening. The HPLC test showed that cadaverine and putrescine were the amines produced in higher amounts by almost all isolates, indicating that the presence of these amines in cheese can be linked to the presence of high counts of Enterobacteriaceae. 44 isolates produced low amounts of histamine (<300 ppm), and four isolates produced more than 1000 ppm of this amine. Only 9 isolates, belonging to the species Citrobacter freundii, Esch. coli and Raoultella ornithinolytica, appeared to produce tyramine. These data provided new information regarding the decarboxylase activity of some Enterobacteriaceae species, including Pantoea agglomerans, Esch. fergusonii and R. ornithinolytica.

Genotypic and phenotypic diversity of Pediococcus pentosaceus strains isolated from food matrices and characterisation of the penocin operon

Lactic acid bacteria (LAB) are widely used in the food industry. Pediococcus spp. belong to the LAB group and include several species that are essential for the quality of fermented food. Pediococcus pentosaceus is the species that is most frequently isolated from fermented food and beverages but its uncontrolled growth during food fermentation processes can contribute to undesired flavours. Hence, the characterisation of these bacteria at the strain level is of great importance for the quality of fermented products. Despite their importance, misidentification at the species level is common for members of the genus Pediococcus. To clarify the taxonomic relationships among strains, a multilocus sequencing approach was developed for the characterisation of a collection of 29 field strains, 1 type strain and 1 reference strain of P. pentosaceus isolated from food. These strains were also tested for several phenotypic properties of technological interest and for the production of bacteriocins. The chromosomal operon involved in the synthesis of the bacteriocin penocin was also investigated. The present study enabled a good genomic characterisation, identifying 17 sequence types, with an overview of phenotypic characteristics related to different technological abilities, and also provides a thorough characterisation of the operon involved in penocin production.