Marielle Bouix

Tunisian Salvia officinalis L. and Schinus molle L. essential oils: Their chemical compositions and their preservative effects against Salmonella inoculated in minced beef meat

The essential oils (EOs) extracted from the aerial parts of cultivated Salvia officinalis L. and the berries of Schinus molle L. were analysed by gas chromatography-mass spectrometry (GC-MS) and 68 and 67 constituents were identified, respectively. The major constituents were 1,8-cineole (33.27%), beta-thujone (18.40%), alpha-thujone (13.45%), borneol (7.39%) in S. officinalis oil and alpha-phellandrene (35.86%), beta-phellandrene (29.3%), beta-pinene (15.68%), p-cymene (5.43%) and alpha-pinene (5.22%) in S. molle oil. In its second part, the present study was conducted to evaluate the in vitro antimicrobial activity of both studied EOs. For this purpose, paper disc-diffusion method and broth microdilution test were used. The disc-diffusion method showed significant zone of lysis against all the pathogens studied (gram-negative and gram-positive bacteria, yeast). These activities remained stable after six months, and decreased approximately by 20% after one year of storage of the EOs at 4 to 7 degrees C. On comparing the efficiency of both EOs, S. officinalis EO exhibited higher antibacterial activity against the majority of strains and especially against Candida albicans (two fold more active according to the inhibition zones values). The minimal inhibitory concentrations (MICs) were reported between 4.5 mg/ml and 72 mg/ml on nutrient broth. The particular chemotype of each EO may be involved in its specific antimicrobial behaviour. Furthermore, the inhibitory effect of these EOs were evaluated against two foodborne pathogens belonging to Salmonella genus, experimentally inoculated (10(3) CFU/g) in minced beef meat, which was mixed with different concentrations of the EO and stored at 4 to 7 degrees C for 15 days. Although the antibacterial activities of both EOs in minced beef meat were clearly evident, their addition had notable effects on the flavour and taste of the meat at concentrations more than 2% for S. molle and 1.5% for S. officinalis. One solution to the above-mentioned problem may be the use of combinations of different food preservation systems. In this context, each of the EOs has been used along with low water activity (addition of NaCl) in addition to low refrigeration temperatures. Results on the Salmonella growth showed that some combinations could be recommended to eliminate germs from minced raw beef. By using this method, a stable and, from a microbiological point of view, safe meat can be produced without substantial loss in sensory quality. Results obtained herein, may suggest that the EOs of S. officinalis and S. molle possess antimicrobial activity, and therefore, they can be used in biotechnological fields as natural preservative ingredients in food and/or pharmaceutical industry.

Tool for quantification of staphylococcal enterotoxin gene expression in cheese.

ABSTRACT Cheese is a complex and dynamic microbial ecosystem characterized by the presence of a large variety of bacteria, yeasts, and molds. Some microorganisms, including species of lactobacilli or lactococci, are known to contribute to the organoleptic quality of cheeses, whereas the presence of other microorganisms may lead to spoilage or constitute a health risk. Staphylococcus aureus is recognized worldwide as an important food-borne pathogen, owing to the production of enterotoxins in food matrices. In order to study enterotoxin gene expression during cheese manufacture, we developed an efficient procedure to recover total RNA from cheese and applied a robust strategy to study gene expression by reverse transcription-quantitative PCR (RT-qPCR). This method yielded pure preparations of undegraded RNA suitable for RT-qPCR. To normalize RT-qPCR data, expression of 10 potential reference genes was investigated during S. aureus growth in milk and in cheese. The three most stably expressed reference genes during cheese manufacture were ftsZ, pta, and gyrB, and these were used as internal controls for RT-qPCR of the genes sea and sed, encoding staphylococcal enterotoxins A and D, respectively. Expression of these staphylococcal enterotoxin genes was monitored during the first 72 h of the cheese-making process, and mRNA data were correlated with enterotoxin production.

Fermentation pH Influences the Physiological-State Dynamics of Lactobacillus bulgaricus CFL1 during pH-Controlled Culture

ABSTRACT This study aims at better understanding the effects of fermentation pH and harvesting time on Lactobacillus bulgaricus CFL1 cellular state in order to improve knowledge of the dynamics of the physiological state and to better manage starter production. The Cinac system and multiparametric flow cytometry were used to characterize and compare the progress of the physiological events that occurred during pH 6 and pH 5 controlled cultures. Acidification activity, membrane damage, enzymatic activity, cellular depolarization, intracellular pH, and pH gradient were determined and compared during growing conditions. Strong differences in the time course of viability, membrane integrity, and acidification activity were displayed between pH 6 and pH 5 cultures. As a main result, the pH 5 control during fermentation allowed the cells to maintain a more robust physiological state, with high viability and stable acidification activity throughout growth, in opposition to a viability decrease and fluctuation of activity at pH 6. This result was mainly explained by differences in lactate concentration in the culture medium and in pH gradient value. The elevated content of the ionic lactate form at high pH values damaged membrane integrity that led to a viability decrease. In contrast, the high pH gradient observed throughout pH 5 cultures was associated with an increased energetic level that helped the cells maintain their physiological state. Such results may benefit industrial starter producers and fermented-product manufacturers by allowing them to better control the quality of their starters, before freezing or before using them for food fermentation.

Effect of inoculum to substrate ratio (I/S) on municipal solid waste anaerobic degradation kinetics and potential

The goal of this study is to evaluate the impact of the inoculum to substrate ratio (I/S) on the anaerobic degradation potential of municipal solid waste (MSW). Reconstituted MSW samples were thus incubated under batch anaerobic conditions and inoculated with an increasing amount of inoculum originating from a mesophilic sludge digester. I/S tested values were 0 (no inoculum added), 0.015, 0.03, 0.06, 0.12, 0.25, 1, 2 and 4 (gVM(inoculum)/gVM(waste)). The results indicate that the apparent maximal rate of dissolved organic carbon accumulation is reached at I/S=0.12. Under this level, the hydrolysis process is limited by the concentration of biomass and can thus be described as first order kinetics phenomena with respect to biomass for I/S ratios below 0.12. The maximum methane production rate and the minimal latency are reached at a ratio of 2. In addition to that, both methane signature and ARISA show a shift in the methanogenic populations from hydrogenotrophic to acetoclastic.

Evaluation of PCR-DGGE methodology to monitor fungal communities on grapes.

Aims:  Some fungi present on the surface of grapes may have a negative effect on the quality of wine. The aim of this study was to evaluate PCR‐denaturing gradient gel electrophoresis (PCR‐DGGE), for the establishment of fungal community profiles from grapes, in order to monitor fungi potentially involved in wine defects.

Dynamic analysis of Lactobacillus delbrueckii subsp. bulgaricus CFL1 physiological characteristics during fermentation

This study aimed at examining and comparing the relevance of various methods in order to discriminate different cellular states of Lactobacillus bulgaricus CFL1 and to improve knowledge on the dynamics of the cellular physiological state during growth and acidification. By using four fluorescent probes combined with multiparametric flow cytometry, membrane integrity, intracellular esterase activity, cellular vitality, membrane depolarization, and intracellular pH were quantified throughout fermentations. Results were compared and correlated with measurements of cultivability, acidification activity (Cinac system), and cellular ability to recover growth in fresh medium (Bioscreen system). The Cinac system and flow cytometry were relevant to distinguish different physiological states throughout growth. Lb. bulgaricus cells maintained their high viability, energetic state, membrane potential, and pH gradient in the late stationary phase, despite the gradual decrease of both cultivability and acidification activity. Viability and membrane integrity were maintained during acidification, at the expense of their cultivability and acidification activity. Finally, this study demonstrated that the physiological state during fermentation was strongly affected by intracellular pH and the pH gradient. The critical pHi of Lb. bulgaricus CFL1 was found to be equal to pH 5.8. Through linear relationships between dpH and cultivability and pHi and acidification activity, pHi and dpH well described the time course of metabolic activity, cultivability, and viability in a single analysis.

Characterization of Streptococcus salivarius growth and maintenance in artificial saliva

Aims:  To help gain a better understanding of factors influencing the establishment within the oral cavity of Streptococcus salivarius K12, a commensal oral bacterium, we characterized its behaviour in artificial saliva.

Rapid enumeration of Oenococcus oeni during malolactic fermentation by flow cytometry

The aim of this study was to provide a method to rapidly enumerate Oenococcus oeni cells during malolactic fermentation (MLF). To keep MLF under control, it is important to monitor the growth of the bacteria O. oeni. However, the enumeration of O. oeni using the plate count technique requires a very long incubation time of about 10 days or more, which is not adapted to monitoring MLF in real time.

Assessment of the dynamics of the physiological states of Lactococcus lactis ssp. cremoris SK11 during growth by flow cytometry

Aims:  The aim of this study was to improve knowledge about the dynamics of the physiological states of Lactococcus lactis ssp. cremoris SK11, a chain‐forming bacterium, during growth, and to evaluate whether flow cytometry (FCM) combined with fluorescent probes can assess these different physiological states.

Rapid assessment of Oenococcus oeni activity by measuring intracellular pH and membrane potential by flow cytometry, and its application to the more effective control of malolactic fermentation

The aim of this study is to highlight the changes in the physiological cellular state of Oenococcus oeni during malolactic fermentation (MLF), and to use its cellular parameters to improve existing knowledge of O. oeni behaviour and to more effectively control the performance of the bacteria during MLF in wine. To do this, measurements of intracellular pH, transmembrane potential and vitality were performed using flow cytometry with different fluorescent probes: CFDA-SE and CDCF, DiBAC and CFDA, respectively. The kinetics of the cellular changes in these parameters were determined during MLF in FT80 synthetic medium and in white wine, as were the kinetics of malic acid consumption. pHin measurement throughout the entire growth shows that the pH was equal to the pH of the culture medium during the early stage, increased to pH6 in the exponential phase, and then decreased to equilibrate with the pH of the medium in the late stationary phase. Membrane potential increased in early MLF and then decreased. The decrease in pHin and membrane potential occurred when all of the malic acid was consumed. Finally, we showed that the higher the ΔpH (pHin-pHex) in O. oeni cells was, the shorter the lag phase of the MLF was. To better manage the initiation of MLF in wines, the physiological state of O. oeni cells must be taken into account. These results allow us to understand the sometimes random initiation of MLF in wines inoculated with O. oeni and to suggest ways to improve this control.