Healthy grapes have a complex microbial ecology including filamentous fungi, yeasts and bacteria. The variety and population of microorganisms present on the grape surface will depend on many factors. However, high sugar concentrations on the berry surface of damaged grapes will especially favour the increase of undesirable microorganisms, such as acetic acid bacteria and wine spoilage yeasts. These microbes are transferred to the must during processing and can have a profound influence on wine composition, flavour and quality. Populations can increase with extended duration of pre-fermentation operations, such as grape transportation, cold soaking, cold storage of juices or grape dehydration. Winemaking strategies to reduce the population of unwanted microbes usually include the use of sulphur dioxide. With the increasing demand for low sulphur dioxide wines, alternative strategies such as bioprotection are explored.
What is bioprotection?
Bioprotection is a natural method to inhibit undesirable indigenous microorganisms present on the grapes or in the must. By adding living organisms specifically chosen for certain properties, the matrix can be colonised in a controlled manner and limit the predominance of the unwanted microflora.
The chosen bioprotector needs to be able to protect the grapes or must from unwanted microbial growth without altering the matrix composition in an undesirable way. The organism needs to be a 1) high-quality strain of 2) oenological origin and 3) have low fermentation activity at the inoculated dose. Other than that, the product needs to be robust to non-rehydration.
Several studies have identified two oenologically derived non-Saccharomyces yeast strains as effective bioprotectors: Torulaspora delbrueckii and Metschnikowia pulcherrima. These strains are ideal for oenological bioprotection due to the following properties:
- M. pulcherrima produces a naturally occurring antimicrobial compound, pulcherrimin, which inhibits the growth of various undesirable microorganisms, such as Brettanomyces bruxellensis and Botrytis cinerea.1,2
- M. pulcherrima scavenge iron which is an essential growth factor for other microorganisms.1
- Both T. delbrueckii and M. pulcherrima tolerate cold conditions well and are thus suitable for use during cold maceration and cold storage in the pre-fermentative stages.3
- The bioprotective actions of both T. delbrueckii and M. pulcherrima do not affect S. cerevisiae activity and studies have shown that S. cerevisiae remains unaffected by the bioprotectors under normal conditions.4 However, at low temperatures, the bioprotectors can inhibit unwanted activity by S. cerevisiae and therefore delay the onset of fermentation (for instance during cold maceration).3
The selected strains have minor effects on the composition of the grapes or must and the effects reported are considered to be more advantageous than disadvantageous:
- T. delbrueckii has been associated with the production of thiol compounds when inoculated sequentially.5
- M. pulcherrima is known to have high beta-glucosidase activity and ester production and may therefore have a positive impact on wine aroma.6
Laffort® offers two bioprotection solutions suitable for oenological applications:
- ZYMAFLORE® ÉGIDETDMP is a formulation consisting of the two strains, delbrueckii and M. pulcherrima.
- ZYMAFLORE® KHIOMP is a specific strain of the pulcherrima species.
ZYMAFLORE® ÉGIDETDMP is particularly suitable as part of a sulphur dioxide reduction strategy. Limiting sulphur dioxide addition during winemaking poses several risks of which the growth of unwanted microorganisms is a major potential implication. The growth of unwanted microorganisms during the pre-fermentative stages will increase the microbiological pressure of the must and prevent the establishment of the inoculated S. cerevisiae yeast. This will significantly delay the onset of fermentation and consequently, the juice will be at risk of oxidation and spoilage. Remedial actions such as reducing the unwanted microbial population and reinoculation with the desired yeasts will further delay fermentation and result in additional costs.
The addition of a bioprotector shortly after harvest (either by direct addition or through contact with treated winery equipment) will significantly reduce the risk of unwanted microbial growth and increase the chances of a successful inoculation and onset of fermentation with the specific chosen S. cerevisiae yeast strain.
ZYMAFLORE® KHIOMP is especially well suited for the oenological bioprotection in low-temperature situations. Pre-fermentative processes such as cold soaking and cold stabulation will require low microbial activity for an extended amount of time. The growth of unwanted microorganisms during these processes will interfere with must clarification and, again, pose the risk of an unsuccessful alcoholic fermentation.
By adding ZYMAFLORE® KHIOMP, the medium is colonised by a microorganism with low fermentation capacity and the population sustained for several weeks, thereby preventing unwanted microbiological activity.
Application in the winery
The ZYMAFLORE® products can be added to the harvest and winery equipment to ensure continuous colonisation of contact surfaces for the duration of harvest. This will prevent accumulation of the unwanted microorganisms such as acetic acid bacteria which tend to flourish during this busy time in the cellar. The products can also be added directly to the must or juice during any of the pre-fermentative stages for better microbial control.
Several field trials were conducted during Bordeaux’s 2020 harvest season to test the effectiveness of the ZYMAFLORE® products. Some of the main findings are summarised below:
The harvester and the winery receiving bin were sprayed with ZYMAFLORE® ÉGIDETDMP before contact with the grapes/must, as well as after each unloading. Must samples were taken at the beginning of the day, halfway through the harvest and at the end of the day and analysed using quantitative polymerase chain reaction (qPCR) technology, as well as plated onto culture media, to assess microbial growth.
Results showed that in the absence of bioprotection, mould and indigenous yeast species flourished, while the must sample from the ZYMAFLORE® treatment showed a dramatically reduced population of undesirable moulds and yeasts while established T. delbruekii and M. pulcherrima populations were evident.
Rinse water trial
Harvest bins were rinsed with ZYMAFLORE® ÉGIDETDMP inoculated water after each unloading. Water samples were collected after rinsing and analysed using media plating and qPCR technology. In the absence of ZYMAFLORE® ÉGIDETDMP mould and non-Saccharomyces yeast species were present, while the treated water was populated with only the two species present in the product.
Juice was kept on the lees for 10 days at 4°C with the aim to maximise the extraction of beneficial compounds and aromatic precursors located in the solids. For this application, ZYMAFLORE® KHIOMP was tested and compared to a treatment to which no bioprotection was added. Results showed that 36% of yeast species in the ZYMAFLORE® KHIOMP-treated juice consisted of S. cerevisiae, while the remainder was M. pulcherrima. The population composition of the control sample (no bioprotection) showed that around 90% of the microflora present at the end of stabulation were indigenous S. cerevisiae species.
Results from the ZYMAFLORE® trials are promising as the use of the products was accompanied by a limited growth of spoilage microbiota. Bioprotection could therefore be used as a tool to successfully manage microbial populations on the winery equipment and in the musts, possibly serving as a credible alternative to sulphites.
During harvest, microorganisms naturally present on the grapes can rapidly multiply increasing the risk of premature fermentation, spoilage and the formation of undesirable compounds. Certain pre-fermentative processes such as cold soaking will support the growth of unwanted microbial populations. Bioprotection is a natural method to inhibit undesirable indigenous microorganisms present on the grapes or in the must. By adding living organisms specifically chosen for certain properties, the matrix can be colonised in a controlled manner and limit the predominance of the unwanted microflora.
- Sipiczki, M., 2006. Metschnikowia strains isolated from botrytized grapes antagonize fungal and bacterial growth by iron depletion. Applied and Environmental Microbiology 72(10), 6716 – 6724.
- Morata, A., Loira, I., Escott, C., del Fresno, J.M., Bañuelos, M.A. & Suárez-Lepe, J.A., 2019. Applications of Metschnikowia pulcherrima in wine biotechnology. Fermentation 5(3), 63.
- Coulon, J., Nazaris, B. & Seabrook, A., 2019. Low SO2 winemaking: Bio-protection for microbial control pre-fermentation. Wine and Viticulture Journal 34(3), 23 – 26.
- Csutak, O., Vassu, T., Avram, I., Stoica, I. & Cornea, C.P., 2013. Antagonistic activity of three newly isolated yeast strains from the surface of fruits. Food Technology and Biotechnology.
- Renault, P., Coulon, J., Moine, V., Thibon, C. & Bely, M., 2016. Enhanced 3-sulfanylhexan-1-Ol production in sequential mixed fermentation with Torulaspora delbrueckii/Saccharomyces cerevisiae reveals a situation of synergistic interaction between two industrial strains. Frontiers in Microbiology 7 (Mar), 1 – 10.
- Parapouli, M., Hatziloukas, E., Drainas, C. & Perisynakis, A., 2009. The effect of Debina grapevine indigenous yeast strains of Metschnikowia and Saccharomyces on wine flavour. Journal of Industrial Microbiology and Biotechnology 37(1), 85.
– For more information, contact Morné Kemp at firstname.lastname@example.org.