Screening brettanomyces
Methodology for the Screening and Isolation of Brettanomyces
in the Grape: plot-scale application
Equipment and Methods
Screening Procedure - Chosen selectivity criteria
The purpose of this procedure was to promote development of the yeast, Brettanomyces (probably not the major contaminant within the ecological
niche investigated) at the expense of the other micro-organisms present in order to identify and screen without being hampered by far more invasive genera.
A study of the literature highlighted a certain number of physiological characteristics, which we have used throughout our procedure in order
to target the Brettanomyces genus and distinguish it from the other species present.
- The use of an antibacterial agent must be considered in order to prevent the proliferation of bacteria (acetic, lactic and others): chloramphenicol possesses a broad spectrum of activity against bacteria as well as fungistatic properties, which could hamper the growth of Brettanomyces.
- Unlike most non-Saccharomyces yeasts found in grapes (including apiculated yeasts), Brettanomyces is highly resistant to ethanol and its growth is not inhibited in a liquid medium at ethanol concentrations below 13% vol/vol. (Medawar 2003).
- Furthermore, the Brettanomyces yeast is actidione-resistant, unlike the Saccharomyces genus, which is subject to proteosynthesis pathway inhibition (Barnett et al. 1990).
- Moreover, under aerobic or semi-aerobic conditions, Brettanomyces is capable of synthesising significant quantities of acetic acid from glucose or ethanol, thus confirming the "acidifying" properties of this yeast (Ciani et Ferraro 1997).
- Finally, Brettanomyces is capable of producing considerable quantities of 4-ethylphenols, which generate unsavoury animal odours (sweat, urine, horse dung, wet leather, etc.) from p-coumaric acid (which is naturally present in grape must and then wine), whereas other micro-organisms present in a vine-growing environment have a very slight aptitude, if any at all, for this conversion into volatile phenols. (Chatonnet et al. 1992, Dias et al. 2003).
Sampling: samples of grapes on the vine and sample preparation
Samples of bunches or several bunches (between 100 g and 300 g) are directly collected under aseptic conditions into sterile sampling bags using cutting tools cleaned with alcohol.
For each sample, the grape is crushed directly in the sterile bag and the stalks are removed under optimal sterile conditions. Each sachet thus obtained is then weighed.
For each plot considered and sampling date, a "weight of grape sample - volume of juice obtained after "pressing" correlation is determined from samples of control bunches.
Figure 1: Grape sample in a sterile sampling bag.
Need for an enrichment phase
Generally speaking, the grape microflora comprises a very small quantity of micro-organisms that are found in the berry film coating.
Thus a low to very low cell concentration cannot be detected simply by directly spreading on a selective medium (the limit being 1 cell per mL of must after pressing). The need for an enrichment phase (cell proliferation / population increase) is easy to understand. This must be sufficiently long in order to reach detectable population levels, regardless of the initial population.
In addition, the physiological condition of the yeasts on the surface of the grape berries does not guarantee direct growth by spreading on an agar medium. We refer to the concept of Viable, Non-Cultivable Cells (VNC), already mentioned by Millet and Lonvaud-Funel (2000) concerning micro-organisms other than bacteria. From now on, an activation stage in a liquid medium is essential. The enrichment phase could also adopt this role.
We believe that this microbial enrichment (accompanied by activation) must be carried out in order to detect the yeasts present. To do this, each sachet of grape juice obtained after "pressing" the samples of bunches is then made up with a specific volume (depending on the volume of juice obtained) of a concentrated, selective solution vis-à-vis Brettanomyces satisfying the afore-mentioned selection criteria. The bags are then left without stirring at a temperature of 20 to 30°C for what we consider to be an optimal period, namely 2 months (give or take 2 weeks) in order to obtain the most relevant results regarding the presence or absence of yeast in the grapes.
Figure 2: Sample during the selective enrichment process
Control and confirmation phases
After 2 months' enrichment in sterile sachets, the samples are processed in order to conclude whether or not Brettanomyces is present. Several stages are then considered.
- Observations beneath the microscope
Initially, examination beneath the microscope can detect the development of yeast and sort between negative samples (no micro-organism visible) and probably positive (presence of microflora indicative of Brettanomyces type morphology) or positive samples (typical morphologies).
Figure 3: Appearance beneath the microscope (x400) of a sample at the start of the enrichment process.
Figure 4: Appearance beneath the microscope (x400) of a sample at the end of the enrichment process: development of yeast with Brettanomyces probably present .
- Confirmation culture on selective media
As morphology is not a conclusive criterion in itself for confirming whether or not yeast belonging to the Brettanomyces genus is present, the samples have to undergo another control procedure.
Samples presenting with any signs of yeast/microbial development at the end of liquid enrichment are spread on a selective, solid medium, which tests for the acidifying properties (acidity indicator) of any yeasts present, on the one hand, and their ability to convert coumaric acid ("sniffing" to detect the formation of 4-ethylphenol) on the other hand.
Spreading on gel allows isolated colonies to be collected and clonal purifications to be carried out on the colonies testing positively to the various selection criteria. Isolation stage in addition to the "detection" approach.
- Genetic test
Genetic tests were also carried out on a certain number of colonies isolated following our complete, applied approach to samples collected from the Buzet vineyard. Specific Brettanomyces PCR identifications (method described by Trevor, Phister and Mills 2003) were thus used to confirm the presence of isolated strains belonging to the Brettanomyces bruxellensis species, as suspected.
From now on, we validate our approach from a specific Brettanomyces detection perspective and the option of isolating strains belonging to this species.
(1) Pascal Barbin, Pierre Strehaiano, Patricia Taillandier - (2) Jean-François Gilis
(1) Chemical Engineering, Bioprocesses and Microbial Systems Laboratory
(2) Oenodev
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