Bioprotection as an alternative to sulphur dioxide in the pre-fermentation phase

Additives have been used in the food industry for many years to prevent spoilage and extend shelf life. These chemical additives are controversial, and their use must be reduced in the face of societal demand. In oenology, this applies in particular to sulphur dioxide (SO₂). There has been recent research on bioprotection as an alternative to sulphite addition in the pre-fermentation phase. This technical article discusses the many advantages of using bioprotection agents.

Background

Many alternatives to sulphur dioxide (SO₂), both physical and chemical, are available on the market or are currently under trial.¹ Among them, one solution is bioprotection by the addition of living microorganisms. This practice, already used in the food sector, involves adding microorganisms capable of colonising the medium. Their presence limits or even inhibits the growth of other undesirable microorganisms without impairing the product’s sensory properties. In oenology, recent research has focused on the detailed impact of using bioprotection as an alternative to SO₂ during the pre-fermentation stages of winemaking.

Competition for space in the grape must

In 2017, three protocols were studied using Merlot:

• Bioprotection (BP) applied at 5 g/hL (without SO₂ addition),
• SO₂ applied at 5 g/hL (50 ppm),²
• Control (Ø) without SO₂.

The bioprotection used (in the form of ADY) was a mixture (50/50) of Torulaspora delbrueckii and Metschnikowia pulcherrima. The manufacturer’s recommendations for rehydration of the bioprotection agent were followed. It was applied by spraying it directly on the grapes.

Three samples were taken during the pre-fermentation phase at 10°C: filling the tank (vatting) and then after 24 hours and 48 hours of cold soaking. Analysis using metabarcoding and high-throughput sequencing was used to characterise the microbial biodiversity of the grape must and determine the relative abundance of different genera and species within the fungal population (Figure 1).

The species used for bioprotection represented an average of 50% of the microflora in the grape must. The relative abundance of T. delbrueckii (light blue) increased during cold soaking, while the reverse was true for M. pulcherrima. The strong presence of these two strains limits the space available for undesirable microorganisms such as Hanseniaspora, Aspergillus and Aureobasidium in the must. The same observation was made with other red Bordeaux musts.³ In addition, bioprotection limits the early establishment of native strains of Saccharomyces cerevisiae, in contrast to the other two protocols. Similar results were also observed in white must using different bioprotection products.

FIGURE 1. Relative abundance (%) of fungal populations in a 2017 Merlot must.⁴

Oxygen consumption by bioprotection

Yeasts consume oxygen as part of their metabolism. The use of bioprotection at a rate of 5 g/hL, corresponding to a concentration in the order of 2 × 10⁶ cells/mL, leads to consumption of dissolved O₂ in the must, as shown by initial trials with whites.⁵ O₂ was consumed more rapidly in the presence of bioprotection (BP), in contrast to the SO₂-free control (Ø), where O₂ consumption is likely due to the activity of polyphenol oxidases. Bioprotection maintained significantly higher glutathione (GSH) concentrations at the end of alcoholic fermentation compared with the control (Figure 2B). This antioxidant compound is naturally present in must and is also synthesised by yeast during alcoholic fermentation. In addition, the presence of bioprotection microorganisms seems to limit must browning (visual assessment) (Figure 2A). Further investigations⁶ have demonstrated that O₂ consumption could be linked to not only the species but also the yeast strain used for bioprotection. Thus, Metschnikowia pulcherrima has an Oxygen Consumption Rate (OCR) significantly greater than the other species (Figure 2C). This means that it consumes O₂ more quickly than other species. In addition, within the same species (e.g., L. thermotolerans), OCR values vary significantly from one strain to another. This ability to consume O₂ could explain the decrease in populations of acetic acid bacteria observed when using bioprotection.⁷

(A)

(B)

(C)

FIGURE 2. O₂ consumption by bioprotection. (A) Sémillon must; (B) [GSH] in Sémillon musts and wines; (C) mean OCR by species (grape juice). BP: 5 g/hL bioprotection; Ø: without SO₂; SO₂: 5 g/hL. ANOVA (p-value ≤ 0.05).

Aromatic profile and sensory impact of bioprotection

In addition to their use at a low dose for bioprotection, non-Saccharomyces yeasts are marketed for their biotechnological properties: they can limit the production of volatile acidity, enhance the fruity aroma of wines, or increase their acidity in the context of climate change. To this end, they are applied at high doses compatible with their contribution to the fermentation process (15 – 30 g/hL) in co-inoculation or sequential inoculation with selected strains of S. cerevisiae (to ensure full completion of the alcoholic fermentation).

Here, the aim was to study the chemical and sensory impact of different non-Saccharomyces yeasts used as low-dose bioprotection agents or applied at high doses in sequential inoculation with S. cerevisiae. The aroma compounds in the wines were analysed and then separated according to the protocol for the application of non-Saccharomyces yeasts (Figure 3A).

The results show that wines from sequential inoculation correlate with higher alcohol acetates. In contrast, those used for bioprotection (with inoculation at a lower dose and without looking for fermentation activity) are correlated with fatty acid ethyl esters. From a sensory point of view, there is a marked impact on fruit perception in young Merlot wines, with wines from sequential inoculation being the most intense, followed by wines obtained using bioprotection, which in turn are more intense than the control wine.

FIGURE 3. Principal Component Analysis of Merlot wines with different applications of non-Saccharomyces yeasts. Sc5: Control, addition of Saccharomyces cerevisiae at 5 g/hL to grapes; Zα5: T. delbrueckii applied for bioprotection at 5 g/hL to grapes; ZE5: mixture of M. pulcherrima and T. delbrueckii applied for bioprotection at 5 g/hL; Zα30: T. delbrueckii applied on filling the tank at 20 g/hL and addition of S. cerevisiae after a loss of 10 density points (sequential inoculation); ZE30: M. pulcherrima and T. delbrueckii applied on filling the tank at 20 g/hL an addition of S. cerevisiae after a loss of 10 density points (sequential inoculation).

In another experiment, sensory analyses were carried out on these wines after 18 months of bottle ageing. The wines made using bioprotection were not sensorially different from those made without SO₂ but did differ from those made with sulphite additions. Nevertheless, the “fresh blackcurrant” descriptor in wines made with bioprotection was scored as more intense than in wines made with sulphite additions.

In conclusion, using non-Saccharomyces yeasts for bioprotection is a promising alternative to sulphur dioxide in the early stages of winemaking, provided the grapes are healthy. All the results indicate that bioprotection offers:

• Partial protection against oxidation phenomena by limiting early browning of musts through consumption of dissolved O₂, thus preserving GSH concentrations in white wines.
• Antimicrobial properties, limiting the relative abundance of certain fungal populations in grape must by competing for space and limiting populations of acetic acid bacteria.
• Chemical and sensory properties, characterised by the production of fatty acid ethyl esters, enhancing fruit perception in young wines.
• Sensory properties after bottle ageing, enhancing the “fresh blackcurrant” score.

References

1. Lisanti, M. T., Blaiotta, G., Nioi, C., & Moio, L. (2019). Alternative Methods to SO₂ for Microbiological Stabilization of Wine. Comprehensive Reviews in Food Science and Food Safety, 18(2), 455‑479. https://doi.org/10.1111/1541-4337.12422.
2. Windholtz, S., Vinsonneau, E., Farris, L., Thibon, C., & Masneuf-Pomarède, I. (2021). Yeast and Filamentous Fungi Microbial Communities in Organic Red Grape Juice: Effect of Vintage, Maturity Stage, SO₂, and Bioprotection. Frontiers in Microbiology, 12, 748416. https://doi.org/10.3389/fmicb.2021.748416.
3. Windholtz, S., Dutilh, L., Lucas, M., Maupeu, J., Vallet-Courbin, A., Farris, L., Coulon, J., & Masneuf-Pomarède, I. (2021). Population Dynamics and Yeast Diversity in Early Winemaking Stages without Sulfites Revealed by Three Complementary Approaches. Applied Sciences, 11(6), Article 6. https://doi.org/10.3390/app11062494.
4. Windholtz, S., Nioi, C., Redon, P., Masneuf-Pomarede, I., & Thibon, C. (2021). Does bioprotection by adding yeasts present antioxidant properties? [Poster]. 8th edition of Macrowine Virtual Congress. https://www.infowine.com/intranet/libretti/0/19546-38%20Sara%20WINDHOLTZ%20Poster%20Macrowine%202021.pdf.
5. Windholtz, S., Nioi, C., Coulon, J., & Masneuf-Pomarède, I. (2023). Bioprotection by non-Saccharomyces yeasts in oenology: Evaluation of O2 consumption and impact on acetic acid bacteria. International Journal of Food Microbiology, 110338. https://doi.org/10.1016/j.ijfoodmicro.2023.110338.
6. Windholtz, S. et al. Non-Saccharomyces yeasts as bioprotection in the composition of red wine and in the reduction of sulfur dioxide. LWT 149, 111781 (2021).
7. Pelonnier-Magimel, E., Windhotz, S., Pomarède, I. M., & Barbe, J.-C. (2020). Sensory characterisation of wines without added sulfites via specific and adapted sensory profile. OENO One, 54(4), Article 4. https://doi.org/10.20870/oeno-one.2020.54.4.3566.

Contributing authors:

Claudia Nioi, Cécile Thibon, Stéphane Bécquet, Emmanuel Vinsonneau,Joana Coulon & Isabelle Masneuf-Pomarède.

This article was originally published on 21 September 2023 in IVES Technical Reviews. It is republished as permitted by the Creative Commons Attribution 4.0 International License of IVES Technical Reviews. Only minor changes were made to suit the style of WineLand Magazine.

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