Optimization of flow cytometry parameters for high-throughput screening of spores of the filamentous fungus Trichoderma reesei.

Flow cytometry (FCM) is a powerful technique still little used to study filamentous fungi due to physical constraints: the hyphae are too large to enter the FCM fluidic system, unless spores can be analyzed at a very early stage of germination. The technique nevertheless has strong potential for the study of these micro-organisms (spore sorting, viability, characterization etc.). This study focused on the investigation of several parameters, ranging from germination and storage conditions of T. reesei spores through to FCM gating, to detect their fluorescence during the first 24 h of germination. Fluorescent spores were first obtained after aerobic germination at 25 °C and monitored over 24 h using FCM, to screen for nine promoters controlling a green fluorescent protein gene. The fluorescence signal (FL1) was then acquired, in addition to the growth characterization of the spores, based on the size signal or Forward Scatter (FSC). They were combined to identify the best candidate(s) from among the nine promoters for the strongest- and earliest-possible fluorescence emission, which resulted in the following ranking: pTEF > pPKI > pGPD > pPDC. There are numerous possible applications of this work, ranging from molecular biology to monitoring fermentation.

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.

Cyclopropanation of unsaturated fatty acids and membrane rigidification improve the freeze-drying resistance of Lactococcus lactis subsp. lactis TOMSC161.

This work aimed at characterizing the biochemical and biophysical properties of the membrane of Lactococcus lactis TOMSC161 cells during fermentation at different temperatures, in relation to their freeze-drying and storage resistance. Cells were cultivated at two different temperatures (22 and 30 °C) and were harvested at different growth phases (from the middle exponential phase to the late stationary phase). Bacterial membranes were characterized by determining the fatty acid composition, the lipid phase transition, and the membrane fluidity. Cultivability and acidification activity losses of L. lactis were quantified after freezing, drying, and 3 months of storage. The direct measurement of membrane fluidity by fluorescence anisotropy was linked to lipid composition, and it was established that the cyclopropanation of unsaturated fatty acids with concomitant membrane rigidification during growth led to an increase in the freeze-drying and storage resistance of L. lactis. As expected, cultivating cells at a lower fermentation temperature than the optimum growth temperature induced a homeoviscous adaptation that was demonstrated by a lowered lipid phase transition temperature but that was not related to any improvement in freeze-drying resistance. L. lactis TOMSC161 was therefore able to develop a combined biochemical and biophysical response at the membrane level during fermentation. The ratio of cyclic fatty acids to unsaturated fatty acids (CFA/UFA) appeared to be the most relevant parameter associated with membrane rigidification and cell resistance to freeze-drying and storage. This study increased our knowledge about the physiological mechanisms that explain the resistance of lactic acid bacteria (LAB) to freeze-drying and storage stresses and demonstrated the relevance of complementary methods of membrane characterization.

Genome Sequence of the Lactic Acid Bacterium Lactococcus lactis subsp. lactis TOMSC161, Isolated from a Nonscalded Curd Pressed Cheese.

Lactococcus lactis is a lactic acid bacterium used in the production of many fermented foods, such as dairy products. Here, we report the genome sequence of L. lactis subsp. lactis TOMSC161, isolated from nonscalded curd pressed cheese. This genome sequence provides information in relation to dairy environment adaptation.

Cell growth and resistance of Lactococcus lactis subsp. lactis TOMSC161 following freezing, drying and freeze-dried storage are differentially affected by fermentation conditions.

AIMS: To investigate the effects of fermentation parameters on the cell growth and on the resistance to each step of the freeze-drying process of Lactococcus lactis subsp. lactis TOMSC161, a natural cheese isolate, using a response surface methodology. METHODS AND RESULTS: Cells were cultivated at different temperatures (22, 30 and 38°C) and pH (5·6, 6·2 and 6·8) and were harvested at different growth phases (0, 3 and 6 h of stationary phase). Cultivability and acidification activity losses of Lc. lactis were quantified after freezing, drying, 1 and 3 months of storage at 4 and 25°C. Lactococcus lactis was not damaged by freezing but was sensitive to drying and to ambient temperature storage. Moreover, the fermentation temperature and the harvesting time influenced the drying resistance of Lc. lactis. CONCLUSIONS: Lactococcus lactis cells grown in a whey-based medium at 32°C, pH 6·2 and harvested at late stationary phase exhibited both an optimal growth and the highest resistance to freeze-drying and storage. SIGNIFICANCE AND IMPACT OF THE STUDY: A better insight on the individual and interaction effects of fermentation parameters made it possible the freeze-drying and storage preservation of a sensitive strain of technological interest. Evidence on the particularly damaging effect of the drying step and the high-temperature storage is presented.

Rapid enumeration of Oenococcus oeni during malolactic fermentation by flow cytometry.

AIMS: 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. METHODS AND RESULTS: Flow cytometry (FCM), in combination with several fluorescent probes, is a rapid method for counting large numbers of bacterial cells. However, probes based on fluorescein [FDA, carboxyfluorescein diacetate (cFDA)] did not give good results for O. oeni. For the first time, we propose using the BacLight™ kit for enumeration of O. oeni, and we compare the results with three methods: plate count and FCM, in combination with either fluorescein probes or the BacLight™kit. The last method provides a perfect correlation with the plate count method. CONCLUSIONS: FCM coupled with the Baclight™ kit makes it possible to count O. oeni cells during MLF with a perfect correlation with the plate count method. SIGNIFICANCE AND IMPACT OF THE STUDY: The result is obtained in 20 min vs 10 days with the reference method which will be very useful for wine microbiologists. Moreover, it should be emphasized that FDA/cFDA staining is not recommended because it can lead to an erroneous count during the latency period or at the end of growth due to the variation of intracellular pH in the O. oeni cells during growth.

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. METHODS AND RESULTS: Cellular viability was assessed using double labelling with carboxyfluorescein diacetate and propidium iodide. FCM makes it possible to discriminate between three cell populations: viable cells, dead cells and cells in an intermediate physiological state. During exponential and stationary phases, the cells in the intermediate physiological state were culturable, whereas this population was no longer culturable at the end of the stationary phase. CONCLUSIONS, AND IMPACT OF THE STUDY: We introduced a new parameter, the ratio of the means of the fluorescence cytometric index to discriminate between viable culturable and viable nonculturable cells. Finally, this work confirms the relevance of FCM combined with two fluorescent stains to evaluate the physiological states of L. lactis SK11 cells during their growth and to distinguish viable cells from viable but not culturable cells.

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. METHODS AND RESULTS: Streptococcus salivarius K12 was grown in artificial saliva complemented with a representative meal, under oral pH and temperature conditions. Exponential growth phase was characterized by a high specific growth rate (2.8 h(-1)). During maintenance phase, an uncoupling between growth and lactic acid production occurred, which allowed maintaining viability (95%), intracellular pH (6.6) and membrane polarisation (95%), and thus proton motive force. However, in late stationary phase, viability (64%) and vitality were degraded as a result of lower synthesis of energetic and glycogen-related proteins as compared to a richer medium. CONCLUSIONS: Streptococcus salivarius was able to rapidly grow in complemented artificial saliva. Nevertheless, a degradation of its physiological state was observed in late-stationary phase. SIGNIFICANCE AND IMPACT OF THE STUDY: This work demonstrates, for the first time, that artificial saliva was a convenient medium that permitted Strep. salivarius to grow in oral conditions (physico-chemical environment, addition of meals) but not to maintain cellular viability and vitality in starvation conditions.

Vinegar production from Togolese local variety Mangovi of Mango mangifera indica Linn. (Anacardiaceae).

The present study aimed to access for the physiochemical parameters of vinegar production through Togolese local variety Mangovi of mango Mangifera indica juice fermentation. The juice was fermented successively by Saccharomyces cerevisisae and acetic bacteria. The levels of ethanol and acetic acid in the juice during the production of vinegar were monitored by gas chromatography and titrimetry methods, respectively. The physiological state of the yeast Saccharomyces cerevisiae L2056 was determined by flow cytometry using a dual fluorescent labeling of diacetate carboxy-fluorescein (CFDA) and propidium iodide. The results indicated that 200 mL of mango juice, sugar content 20 Brix, set in alcoholic fermentation with 10(6) yeast cells produced 22.4 g L(-1) ethanol in 72 h. Acetic fermentation transformed 93% of this ethanol to acetic acid in 288 h. Twenty-four hours after the beginning of alcoholic fermentation, 91% of cells were viable, 8.85% were stressed and 0.05% died. After 24 h of acetic fermentation, viable, stressed and dead cells were 45, 12 and 39%, respectively; corresponding to the passage of acetic vinegar level from 0.9 to 2.1 degrees. At the end of the acetic fermentation, dead cells were estimated to 98% at and acetic acid to 4.7 degrees. Using consecutive fermentations is suitable technique for vinegar production from mango juice. The application of the present results may contribute to avoid fruits post harvest losses.

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. METHODS AND RESULTS: A fragment of the beta-tubulin gene was amplified from filamentous fungi and yeasts described from grapes and analysed using two different denaturing gradient gels to constitute a reference database. The use of beta-tubulin sequences instead of ITS rDNA in PCR-DGGE showed a progress in the discrimination of these fungal species but comigration problems were still observed. The technique was then applied on grape samples. The profiles counted up to 10 bands of which half corresponded to species which were not recorded in the reference database. CONCLUSION: PCR-DGGE represents a useful tool to compare environmental samples for the study of the dynamics of fungal communities, but comigrations represent a limit in its use to describe the species present. SIGNIFICANCE AND IMPACT OF THE STUDY: A better knowledge of the fungal diversity on grapes, particularly species responsible for wine defect, is necessary to develop accurate molecular detection tools.

Analysis of fungal diversity of grapes by application of temporal temperature gradient gel electrophoresis - potentialities and limits of the method.

AIMS: Fungi could be responsible for several problems in wines but the fungal ecosystem of grapes remains little known. The use of traditional methods does not allow to describe quickly this ecosystem. Therefore, we need to improve the knowledge about these fungi to prevent defects in wine. This study aims at evaluating the potentialities of the temporal temperature gradient gel electrophoresis (TTGE) method to describe the fungal ecosystem of grapes. METHODS AND RESULTS: The internal transcribed spacer (ITS) region was amplified and analysed using TTGE. A reference database of 56 fungal species was set up to evaluate the discrimination power of the method. The database was used for the direct identification of the fungal species present in complex samples. The sensitivity of the method is below 10(4) spores per ml. CONCLUSIONS: This method allows to describe the fungal diversity of grapes, but does not always allow to directly identify all fungal species, because of the taxonomic resolution of the ITS sequences. However, this identification strategy is less time consuming than traditional analysis by cloning and sequencing the bands. SIGNIFICANCE AND IMPACT OF THE STUDY: With this method, it will be possible to compare the fungal species present in different vineyards and to connect the presence of some fungi with particular defects in wine.

Physiological significance of the cytometric distribution of fluorescent yeasts after viability staining.

This article describes a new method for the early detection of alcoholic fermentation arrest. This methodology is based on the flow cytometric assessment of Saccharomyces cerevisiae yeasts stained with a carboxyfluorescein diacetate fluorescent viability probe. Multicomponent analysis of viable cell distribution constitutes a promising new tool to describe physiological and dynamic changes to heterogeneous viable populations during alcoholic fermentation, through its ability to discriminate between successful processes and those ending prematurely. This framework, which is based on the comparison of cytometric histogram descriptors' combinations that can be related to simple physiological significance comparison, quickly and simply, allows testing yeasts for their fermentation ability and can be used to detect any kind of viability loss so that fermentation arrest can be avoided.

Electrophoretic study of the macromolecular compounds excreted by yeasts: application to differentiation between strains of the same species.

The macromolecular compounds excreted during growth by yeasts as exocellular fractions were studied and found to contain a high proportion of carbohydrates. On the basis of the specificity of these fractions, an attempt was made to distinguish between nine strains of a single species of yeast-Saccharomyces cerevisiae. Each strain came from a different source. The electrophoretic patterns of the exocellular fractions tested made it possible to distinguish between the nine strains, on the basis of Lodder's criteria for the morphological and physiological identification of yeasts. We considered that strains giving exocellular fractions distinguishable by electrophoresis belonged to distinct clones. Consequently, this technique enables us to go further than diagnosis of the species.