Aller au contenu  Aller au menu principal  Aller à la recherche

UMR Procédés Alimentaires et Microbiologiques

bandeau_umr-pam.jpg

Food Wine Microbiology and Stress (VAlMiS)

valmis schema en

 


Research topics

The VAlMiS team (Food Wine Microbiology and Stress) specialises in studying vine and wine microorganisms and characterising host cell - microbiota interactions.

Topic 1: Vine and wine microorganisms

I. Adaptation of lactic acid bacterium Oenococcus oeni in wine

O. oeni is the main lactic acid bacterium involved in malolactic fermentation (MLF) in wine. In addition to modifying the acidity due to malic acid decarboxylation, this second fermentation strongly influences the organoleptic quality of wine, especially red wine. The extreme physico-chemical conditions in wine (low pH and temperature) associated with the presence of biological competitors (yeast and spoilage bacteria) is detrimental to the development and survival of O. oeni and consequently the successful completion of MLF. Our aim is to improve understanding of the mechanisms involved in the adaptation of O. oeni in wine and to optimise its preservation in optimal conditions in this harsh environment to improve MLF.


A. Culture method and malolactic fermentation: Oenococcus oeni biofilms
(Collaboration: PCAV PAM Joint Research Centre; Micalis, B2HM, Massy, France; ISVV, Bordeaux, France)

O. oeni biofilms formed on stainless steel or wood (figure 1) can be used to trigger MLF and alter the aromas in wine during ageing. This culture method helps optimise survival of the bacteria (improvement in response to environmental stress) and effectively trigger MLF. Tests have been conducted on a laboratory scale but also under winemaking conditions at the experimental centre on the University of Burgundy Franche-Comté wine-growing estate in Marsannay. This work led to a patent being filed.  
The culture of
O. oeni biofilms promotes the production of exopolymeric substances (EPS). Our aim is to characterise the structure and production dynamics of these substances during biofilm formation and their influence on the resistance of the bacterium to environmental stress. A link between this culture method and lysogeny will be studied more closely.

 fig1 valmis en


Figure 1: SEM photo x 35 000 of
O.oeni ATCC BAA-1163 cells in a biofilm on wood.

 

 

B. Molecular basis of stress response

1. Interaction of Lo18 with the cytoplasmic membrane

(Collaboration: ICB, Dijon, France; IBS, Grenoble; Micalis, B2HM, Massy)

The small stress protein (sHsp) Lo18 is essential to maintaining membrane fluidity and dealing with proteins damaged as a result of environmental stress. Lo18 is a dynamic protein that adopts different configurations according to the protein (oligomers) or lipid (dimers) substrates with which it interacts (figure 2A). It has a greater affinity with the membrane in a liquid state in relation to the fatty acid composition. The oligomerization dynamics of this sHsp, as well as Lo18-membrane interactions, have been characterised in situ using atomic force microscopy (figure 2 B and C).

fig2

Screenshot 2

Figure 2: A) Model of the stress response of Oenococcus oeni to ethanol. B) Oligomerization dynamics of sHsp Lo18 from pH 9 to pH 5 observed using HS-AFM (scan speed: 4 frames/s; scan size: 150 nm x 150 nm). The arrows indicate the progression of the dimers in the oligomerization process. The series of images highlights the complete oligomerization process carried out in less than 80s (reversible process). C) Three-dimensional structure of the Lo18 protein reconstructed from the images obtained using cryo-electron microscopy at pH 5.

2. CtsR: coordinator of the stress response
(Collaboration: Biology of gram-positive pathogenic bacteria Laboratory, Institut Pasteur, Paris)

Five different mechanisms control hsp (heat shock protein) gene expression in B. Subtilis. However, the comparative analysis of the O. oeni genome has shown that CtsR is currently the only known gene involved in the stress response in this bacterium of oenological importance. Characterised mainly in B. subtilis, CtsR is a transcriptional repressor involved in controlling the expression of class III genes (clpP and clpC operon). The generation of transcriptional fusions involving promoter regions of hsp genes of O. oeni has confirmed the function of this transcriptional repressor in O. oeni. The analysis of the level of expression of these fusions conducted in a heterologous expression system in B. subtilis has highlighted CtsR-dependent regulation of most O. oenihsp genes, thus underlining the singularity of this lactic acid bacterium with regard to its regulation of stress response. Analysis using comparative genomics indicates that CtsR regulation does not fit the same model as that described in B. subtilis. We are now looking to focus our research on characterising the mechanisms involved in the regulation of CtsR activity in O. oeni

 

C. Implementation of tools for the functional exploration of O. oeni genes

(Collaboration: Biology of gram-positive pathogenic bacteria laboratory, Institut Pasteur, Paris)

O. oeni is particularly resistant to genetic modification techniques commonly used for lactic acid bacteria, thus restricting the functional characterisation of the genes in this bacterium. Until now, the function of these genes had been studied in heterologous expression systems and so the cfa gene, which is expressed under stress conditions, was characterised in E. coli then in Lactococcus lactis. The biochemical characterisation of this CFA synthase has brought to light a probable specificity of this enzyme regarding O. oeni membrane lipids. Particularly long and tedious, the development of genetic tools suitable for this bacterium has led to the development of an electroporation technique using ethanol to increase membrane fluidity, and it is now possible to introduce plasmid vectors into O. oeni. Using genetic tools developed for Lactobacillus plantarum, a vector was adapted for use as an expression vector in O. Oeni, which led to the expression of esterase-encoding genes in O. oeni strain ATCC BAA 1163. The esterase activity detected in the recombinant strains has helped assess the functionality of these genes as well as the impact of their gene products on the aromatic profile of wine during MLF. Based on this new tool, antisense RNA expression has been implemented to modulate in vivo the expression of target genes. Applied to gene hsp18, this knock-down technology has highlighted in vivo the role of Lo18 in the tolerance of this bacteria to an acidic environment and heat stress. 
Thanks to these technological advances in the ability to manipulate the
O. oeni genome, we can continue characterising in vivo the stress response in O. oeni, and in particular the mechanisms involved in the regulation of CtsR activity.

 

II. Microbial ecology 

A. Impact of anthropogenic factors on grape and cellar flora 

Collaboration: Institut für Mikrobiologie und Biochemie Zentrum Analytische Chemie und Mikrobiologie, Hochschule Geisenheim University, Geisenheim, Germany; Department of Biology-Genetics, University of Bari, Italy; PCAV team, PAM Joint Research Centre, Dijon).

We are investigating the effects of various anthropogenic activities (vineyards, winery) on fungal populations from the grapes to the wine. In the vineyards, the impact of phytosanitary protection has been highlighted: lower fungal biodiversity was observed in 3 consecutive vintages produced using organic methods compared with conventional methods (figure 3). In the winery, the fungal populations change significantly after pressing/clarification, but the differences observed in the vineyards remain. Through non-targeted analysis of the wine obtained, hallmarks of chemical and microbiological diversity linked to the method of protecting the vineyards have been highlighted, and we have shown for the first time that the grape berry is a limited source for non-Saccharomyces (NS) yeasts whereas the winery seems to be a major source. Furthermore, the persistence and re-establishment of NS yeast in the must the following year has been observed. This work led us to initiate a new study on the dynamics of indigenous yeast populations in a new winery to understand their establishment and persistence over the following years.

fig4

Figure 3: Distribution of the fungal flora on grape berries from conventional,
organic or Ecophyto (reduction in use of phytosanitary products) vineyards.
The populations were identified using pyrosequencing.

 

B. Interactions between microorganisms or how to control mixed-culture fermentation
(Collaboration: PCAV team, PAM Joint Research Centre, Dijon; Research Unit Analytical BioGeoChemistry, Department of Environmental Sciences, Helmholtz Zentrum München, Ingolstädter Landstr.1, Neuherberg, Germany]

Microorganisms interact with each other during fermentation processes. These interactions are the result of molecular and physiological mechanisms and subsist during both natural and industrial fermentation. In the latter case, complex yeasts have been developed but their use is limited due to a lack of knowledge regarding the mechanisms of interactions occurring during mixed fermentation. The main aims of the research conducted by the VAlMiS team are to improve understanding of these mechanisms of interaction in order to improve control of mixed culture fermentation as well as to improve understanding of the population dynamics in indigenous fermentation.
Using a metabolomic approach, the team has already shown that a microorganism can modulate the metabolism of another microorganism. The next challenge is to understand how a species can change the metabolism of another species. In order to characterise the nature of the interactions, the population dynamics in mixtures will be monitored using multiparametric analysis. The existence of cell communication mechanisms or cell-contact will be investigated and the impact of environmental parameters on the interactions will be studied. For this purpose, the laboratory has acquired expertise in multiparametric analysis of yeasts using flow cytometry.

C. Non-Saccharomyces yeasts: benefits and impact on S. cerevisiae metabolism

The use of yeasts in biotechnology, in particular non-Saccharomyces (NS) yeasts, is a rapidly expanding field of research for the VAlMiS team. NS yeasts, formerly described as being undesirable, are now studied for their oenological and organoleptic potential. Several studies are currently underway to understand the implication of NS yeasts during vinification of which the main objectives are to assess their impact on biodiversity and aromatic complexity.
The first area of research addresses the use of NS yeasts in reducing inputs of exogenous chemicals such as SO
2. Understanding and assessing the impact of NS yeasts is, therefore, essential to control their use in the bio-protection of grape must and thus reduce the concentration of sulphites from an antiseptic and antioxidant point of view.

Screenshot 4

Figure 4: Isolation of non-Saccharomyces
yeasts from Aligoté must (S. Simonin)

 

In addition to studies on the use of NS yeasts as an alternative to sulphites in bio-protection, the second area of research looks at the use of NS yeasts to decrease the concentration of alcohol in wine. In recent years, with global warming, the concentration of sugar in must has been increasing, resulting in wines with a higher alcohol content. Solutions already exist but their use can affect the organoleptic quality of the wine, which is why using NS yeasts through sequential inoculation is being investigated. These yeasts do not have the same ethanol yields, and thus a decrease of nearly 2% in the final ethanol concentration is conceivable. With the perspective of using new technological processes to control processes involving microorganisms, the studies underway aim to improve understanding of the mechanisms involved with these yeasts and the impact of their presence on Saccharomyces cerevisiae metabolism.

Based on the previous two areas, the third area of research looks at the interactions between the yeast Saccharomyces cerevisiae and NS selected for their organoleptic potential in the finished wine. Indeed, laboratory studies have shown that NS can produce aromatic compounds desirable in wine. However, this ability is lost in the presence of S. cerevisiae or NS suffer premature mortality at the start of alcoholic fermentation (AF) due to interactions. The mechanisms involved as well as the nature of the interactions that could occur are described little and poorly understood. The aim is to determine and to characterise these interactions between yeast species during AF in order to provide transferable knowledge for the wine industry.

III. Brettanomyces: method of survival in wine and in the cellar 
(Collaboration: Microflora; Inter-Rhône; BIVB)

Brettanomyces (figure 5) is a spoilage yeast in wine. It produces volatile phenols that spoil the wine and so the industry is looking for means to detect this yeast in particular and to understand its resistance to sulphites.
For the first time, we have studied the link between the quantity of SO
2 and the yeast population in wine and have shown that for the same dose of SO2, the greater the population, the less effective the sulphites. We have detected viable but non-culturable Brettanomyces in wine; they do not produce 4-ethylphenol but can become culturable again and spoil the wine as the sulphite concentration decreases over time. These results show the importance of accurate quantification of Brettanomyces
To respond to the industry’s concerns regarding the presence of
Brettanomyces during ageing, we are studying the evolution over time of the flora present in the cellar (bacteria, total yeast, Brettanomyces). The study concerns 3 cellars (with or without problems with Brettanomyces contamination, using or not yeast starters, and with varying standards of hygiene), to help improve understanding of the composition of the cellar flora and its evolution over time. Brettanomyces will be monitored down to strain level in order to see if problem cellars have more Brettanomyces and/or more resistant strains (particularly to SO2) than cellars with no problems.

Screenshot 1

Figure 5: SEM photo of Brettanomyces cells (Serpaggi, 2011).

Topic 2: Host cell-microbiota interactions

I. Beneficial effect of biofilms on the formulation, preservation, and functions of probiotics
(Collaboration: Micalis, B2HM, Massy, France; PCAV team, PAM Joint Research Centre, Dijon)

Our team specialises in the development of probiotic biofilms (figure 6). We have shown that the biofilm phenotype promotes the establishment and beneficial effects of bacteria in the digestive tract and we have developed edible biofilms of probiotic Lactobacillus bacteria. The objective is to develop a polyoside-based edible film to which probiotic bacteria can adhere and form a biofilm with the aim of producing probiotics that are (i) more resistant to the stressful conditions in the digestive tract, (ii) capable of becoming established in the intestine, and (iii) have optimal functions.

fig6

Figure 6: Development of a Lactobacillus casei P334 biofilm stained with
Cyto9 and observed under a confocal microscope.

II. Molecular dialogue between microorganisms and host cells 

A. Modulation of key processes of innate and adaptive immunity: autophagy  

(Collaboration: Micalis, Probihôte, Jouy-en-Josas, France)

The aim of this topic is to develop innovative, natural or genetically modified probiotic microorganisms that can help intestinal homoeostasis, in particular by stimulating the autophagy process. Autophagy is an intracellular catabolic process essential to the survival of each eukaryotic cell (for more information visit http://cfatg.org/outils/pedagogiques/). If stimulating autophagy or maintaining its function is associated with health benefits and a greater life expectancy, conversely, alterations to this process are associated with ageing as well as numerous conditions such as chronic intestinal inflammatory conditions and diabetes. 
To date, there is very little data on the regulation of autophagic processes by commensal microorganisms of the intestinal microbiota or probiotic microorganisms. The objective of our research is twofold: (1) to understand every aspect of the interactions of commensal bacteria and probiotics, yeasts or the intestinal microbiota in basal conditions (homeostasis) and in pathophysiological situations, and (2) to use natural or genetically modified probiotic bacteria to stimulate autophagy (figure 7) and thus reinforce intestinal homeostasis. The effects of these microorganisms on the autophagy process are characterised in vitro using lines of intestinal epithelial cells and in vivo in mice (germ-free or conventional) and in the zebrafish.

fig7

Figure 7: Stimulation of autophagy in human cells by a strain of Lactobacillus casei (L. casei).
Activation of autophagy monitored using immunofluorescence (left panel) with LC3 protein immunostaining. An increase in the number of LC3 puncta per cell shows activation of autophagy Publication: Al Azzaz et al. manuscript
in preparation

B. Role of HSP GroEL in the interaction and modulation of the immune response by Lactobacillus casei
(Collaboration: Micalis, B2HM, Massy, France; LNC, Dijon, France; CSIC, Valencia, Spain)

The power of probiotic bacteria biofilms to modulate innate intestinal immunity in particular has been characterised and the results published by our laboratory have shown that the formation of biofilms of strains of Lactobacillus not only increases their anti-inflammatory properties but also their resistance to physic-chemical stress mimicking the gastrointestinal tract (figure 8). These results were obtained in vitro on human immunological lines and in vivo in the zebrafish model. It was observed that the molecular shapes responsible for this anti-inflammatory effect were associated with the wall and were also released into the supernatant. We have identified the essential role of the GroEL protein in this attenuation phenomenon of the immune response in vitro. This protein of the HSP (Heat Shock Protein) family is present in greater quantities in biofilm culture supernatants.

Screenshot 3

Figure 8: Colonisation of the intestinal epithelium of zebrafish larvae by Lactobacillus casei observed using transmission electron microscopy.

C. Cell, molecular, and immunological mechanisms involved in the digestive colonisation of Candida albicans and the role of stress factors
(Collaboration: CNRS, RIDI UPR 9022, Strasbourg, France; Institut Pasteur, Unité Biologie et Pathogénicité Fongiques, INRA, USC 2019, Paris, France; Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology-Hans Knoell Institute, Jena, Germany) 

fig9

Candida albicans (C. albicans) is a eukaryotic microorganism belonging to the commensal intestinal (i.e. the microbiota), oral, and vaginal flora in healthy humans (figure 9).

Figure 9: Scanning microscope image showing the interaction of a filamentous form of Candida albicans with Caco-2 line digestive cells

This commensalism results in an equilibrium between the yeast and the host defence systems. The disruption of this equilibrium in weakened patients (with HIV, neutropenia, or cancer, organ recipients or in intensive care) results in intense colonisation of the mucosa promoting the invasion of the epithelial cells, translocation through the epithelial digestive barrier, and haematogenic dissemination of the yeast. This microbial study design is very innovative in the sense that the nature of the gastrointestinal environment in humans will influence its existence in commensal or pathogenic state. The objective of our research is to specify the cellular and molecular mechanisms of interaction of C. albicans with the digestive mucous using in vitro and in vivo study designs. The characterization of the molecules expressed by the yeast in various morphological forms (i.e. planktonic or biofilm) and contributing to the molecular dialogue with the host cell is addressed.

Read more: Food Wine Microbiology and Stress (VAlMiS)

Subcategories

logo Qualiment logo court ASD 
Université de Bourgogne