Giuseppe Comi

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.

Denaturing Gradient Gel Electrophoresis Analysis of the 16S rRNA Gene V1 Region To Monitor Dynamic Changes in the Bacterial Population during Fermentation of Italian Sausages

ABSTRACT In this study, a PCR-denaturing gradient gel electrophoresis (DGGE) protocol was used to monitor the dynamic changes in the microbial population during ripening of natural fermented sausages. The method was first optimized by using control strains from international collections, and a natural sausage fermentation was studied by PCR-DGGE and traditional methods. Total microbial DNA and RNA were extracted directly from the sausages and subjected to PCR and reverse transcription-PCR, and the amplicons obtained were analyzed by DGGE. Lactic acid bacteria (LAB) were present together with other organisms, mainly members of the family Micrococcaceae and meat contaminants, such as Brochothrix thermosphacta andEnterococcus sp., during the first 3 days of fermentation. After 3 days, LAB represented the main population, which was responsible for the acidification and proteolysis that determined the characteristic organoleptic profile of the Friuli Venezia Giulia fermented sausages. The PCR-DGGE protocol for studying sausage fermentation proved to be a good tool for monitoring the process in real time, and it makes technological adjustments possible when they are required.

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.

Culture-Dependent and -Independent Methods To Investigate the Microbial Ecology of Italian Fermented Sausages

ABSTRACT In this study, the microbial ecology of three naturally fermented sausages produced in northeast Italy was studied by culture-dependent and -independent methods. By plating analysis, the predominance of lactic acid bacteria populations was pointed out, as well as the importance of coagulase-negative cocci. Also in the case of one fermentation, the fecal enterocci reached significant counts, highlighting their contribution to the particular transformation process. Yeast counts were higher than the detection limit (>100 CFU/g) in only one fermented sausage. Analysis of the denaturing gradient gel electrophoresis (DGGE) patterns and sequencing of the bands allowed profiling of the microbial populations present in the sausages during fermentation. The bacterial ecology was mainly characterized by the stable presence of Lactobacillus curvatus and Lactobacillus sakei, but Lactobacillus paracasei was also repeatedly detected. An important piece of evidence was the presence of Lactococcus garvieae, which clearly contributed in two fermentations. Several species of Staphylococcus were also detected. Regarding other bacterial groups, Bacillus sp., Ruminococcus sp., and Macrococcus caseolyticus were also identified at the beginning of the transformations. In addition, yeast species belonging to Debaryomyces hansenii, several Candida species, and Willopsis saturnus were observed in the DGGE gels. Finally, cluster analysis of the bacterial and yeast DGGE profiles highlighted the uniqueness of the fermentation processes studied.

Repeatability of surface EMG variables during voluntary isometric contractions of the biceps brachii muscle.

The repeatability of initial value and rate of change of mean spectral frequency (MNF), average rectified values (ARV) and muscle fiber conduction velocity (CV) was investigated in the dominant biceps brachii of ten normal subjects during sustained isometric voluntary contractions. Four levels of contraction were studied: 10%, 30%, 50% and 70% of the maximal voluntary contraction level (MVC). Each contraction was repeated three times in each of three different days for a total of nine contractions/level/subject and 90 contractions per level across the ten subjects. Repeatability was investigated using the Intraclass Correlation Coefficient (ICC) and the standard error of the mean (SEM) of the estimates for each subject. Contrary to observations in other muscles, CV estimates appeared to be very repeatable both within and between subjects. CV showed a small but significant increase when contraction force increased from 10% to 50% MVC but no change for further increase of force. As force increased, MNF showed a slight decrease possibly related to a wider spreading of the CV values. The rate of time decrement of MNF and CV increased with the level of contraction. The normalized decrement (% of initial value per second) was in general higher for MNF than for CV and was more repeatable between subjects at 10% MVC than at 70% MVC. A final observation is that a resting time of 5 minutes may not be sufficient after a contraction at 50% or 70% MVC.

Characterisation of naturally fermented sausages produced in the North East of Italy.

In the Friuli Venezia Giulia region, in the North East of Italy, a traditional fermented sausage is produced without the use of microbial starters. It is characterized at the end of the ripening period by accentuated acidity, slight sourness and elastic, semi-hard consistency. In this study, three fermentations, carried out in different seasons (winter, spring and summer) were followed analyzing the microbiological, physicochemical and sensory aspects of this product. The sausages were characterized by an important microbial activity of lactic acid bacteria and micro/staphylococci that resulted in a product with a final pH of about 5.6-5.7. An interesting aspect was the high number of fecal enterococci that can play an important role in the definition of the organoleptic profile of the final product. No Listeria monocytogenes, Salmonella spp. and Staphylococcus aureus were ever isolated from the raw materials or the fermented sausages during the maturation, underlining the safety of this product. The final water activity of the product was 0.91-0.92. One hundred and fifty lactic acid bacteria were isolated and identified by molecular methods to understand which species were more predominant in the product. Lactobacillus curvatus and Lactobacillus sakei were the most numerous (54 and 64 strains isolated, respectively) and they were the only species common to all three fermentations. A cluster analysis of the profiles obtained from these strains after RAPD-PCR highlighted a population distribution that was fermentation-specific.

Study of the Ecology of Fresh Sausages and Characterization of Populations of Lactic Acid Bacteria by Molecular Methods

ABSTRACT In this study, a polyphasic approach was used to study the ecology of fresh sausages and to characterize populations of lactic acid bacteria (LAB). The microbial profile of fresh sausages was monitored from the production day to the 10th day of storage at 4°C. Samples were collected on days 0, 3, 6, and 10, and culture-dependent and -independent methods of detection and identification were applied. Traditional plating and isolation of LAB strains, which were subsequently identified by molecular methods, and the application of PCR-denaturing gradient gel electrophoresis (DGGE) to DNA and RNA extracted directly from the fresh sausage samples allowed the study in detail of the changes in the bacterial and yeast populations during storage. Brochothrix thermosphacta and Lactobacillus sakei were the main populations present. In particular, B. thermosphacta was present throughout the process, as determined by both DNA and RNA analysis. Other bacterial species, mainly Staphylococcus xylosus, Leuconostoc mesenteroides, and L. curvatus, were detected by DGGE. Moreover, an uncultured bacterium and an uncultured Staphylococcus sp. were present, too. LAB strains isolated at day 0 were identified as Lactococcus lactis subsp. lactis, L. casei, and Enterococcus casseliflavus, and on day 3 a strain of Leuconostoc mesenteroides was identified. The remaining strains isolated belonged to L. sakei. Concerning the yeast ecology, only Debaryomyces hansenii was established in the fresh sausages. Capronia mansonii was initially present, but it was not detected after the first 3 days. At last, L. sakei isolates were characterized by randomly amplified polymorphic DNA PCR and repetitive DNA element PCR. The results obtained underlined how different populations took over at different steps of the process. This is believed to be the result of the selection of the particular population, possibly due to the low storage temperature employed.

An application of PCR-DGGE analysis to profile the yeast populations in raw milk

Four different zones from the Friuli Venezia Giulia region, North East of Italy, were sampled for the study of the yeast bio-diversity in raw milk. Samples were analysed by traditional methods to isolate different yeast strains that were subjected to identification by sequencing the D1–D2 domains of the 26S rRNA gene. Twelve different species of yeast were identified, six of them belonging to the genera Candida and two to the genera Kluyveromyces. The identified strains were then used for the optimization of a method based on polymerase chain reaction and denaturing gradient gel electrophoresis that was used for a direct monitoring of the populations in the samples. Applying the method to the DNA extracted directly from the raw milk samples, new bands appeared in the gel underlining a different bio-diversity in respect to the traditional method. The approach described is a powerful and reliable tool to monitor directly yeast ecology in milk and milk products without the need of traditional isolation and it could be used to follow specific populations to prevent spoilage or to control contamination.

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for the Discrimination of Food-Borne Microorganisms

ABSTRACT A methodology based on matrix-assisted laser desorption ionization-time of flight mass spectrometry of intact bacterial cells was used for rapid discrimination of 24 bacterial species, and detailed analyses to identify Escherichia coli O157:H7 were carried out. Highly specific mass spectrometric profiles of pathogenic and nonpathogenic bacteria that are well-known major food contaminants were obtained, uploaded in a specific database, and made available on the Web. In order to standardize the analytical protocol, several experimental, sample preparation, and mass spectrometry parameters that can affect the reproducibility and accuracy of data were evaluated. Our results confirm the conclusion that this strategy is a powerful tool for rapid and accurate identification of bacterial species and that mass spectrometric methodologies could play an essential role in polyphasic approaches to the identification of pathogenic bacteria.

The late blowing in cheese: a new molecular approach based on PCR and DGGE to study the microbial ecology of the alteration process.

A molecular biology method based on polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) was developed to detect Clostridium spp. in cheese samples suspected of late blowing. Strains of Clostridium spp. and different Lactic Acid Bacteria species, obtained from international collections, were used to determine the experimental conditions for the PCR amplification and DGGE differentiation. DNA extracted directly from cheeses with late blowing symptoms was subjected to PCR and DGGE analysis and traditional agar plating was performed for samples pasteurized and enriched overnight. Moreover, volatile fatty acids were determined for comparison purposes. The PCR-DGGE results were in agreement with the plating performed, and only samples presenting DGGE bands migrating at the same position as Clostridium spp. bands, showed the presence of Clostridium colonies on Reinforced Clostridial Medium plates. Butyric acid contents were high (>100 mg/kg) in the cases of positive DGGE results, underlining the suitability of the protocol for the study of cheese spoilage. The sensitivity of the method is estimated to be 10(4) CFU/g.