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Commercial Non-Saccharomyces Yeast – Interesting Adjuncts to Saccharomyces cerevisiae with Particular Reference to Metschnikowia pulcherrima

Jason Mabbett, Technical Applications Manager, AB Biotek

Commercial Non-<i>Saccharomyces Yeast</i> – Interesting Adjuncts to <i>Saccharomyces cerevisiae</i> with Particular Reference to <i>Metschnikowia pulcherrima</i>

Isolation of pure yeast cultures and the understanding by yeast manufacturers of how to produce them as active dried wine yeast has improved since 1965, when they were first commercially introduced. Saccharomyces cerevisiae became the preferred yeast for fermenting grape juice and must. But why is this?

From a technical perspective the fact that S. cerevisiae, relatively speaking, is an easy yeast to grow, can be stored for long periods in suspended animation and has accessible genetics that can be followed through sexual and asexual cycle is a huge advantage. This has lent itself to use in food and beverage production over millennia.1 With specific regard to winemaking it largely it comes down to the natural ability of S. cerevisiae to dominate during fermentation and their desirable flavor properties.7 This has led to greater control of vinifications, more predictable outcomes and decreases in the risk of spoilage by other microorganisms.

Not content, however, with S. cerevisiae strains sourced from natural or spontaneous ferments, yeast researchers and manufacturers have turned to non-genetically modified methods to modulate fermentation performance, aroma and flavor. Through the use of back-crossing of targeted gene deletion, rare mating, yeast strain isolation and mutagenesis researchers have been able to arm winemakers with additional new tools to address the needs of consumers. For example, inhibiting the development of hydrogen sulfide from yeast has generated cleaner, more aromatic wines. Advances in research related to genetics and molecular biology have had implications for wine science and winemaking by increasing the yeast options available.  

While there has also been the advent of genetically modified (GMO) wine yeasts, prevailing anti-GMO sentiments from both consumers and other global regulatory authorities, have minimized widespread commercial usage despite some attractive attributes. In many respects, it is against this background of increased technological innovation and capabilities (especially from a manufacturing standpoint), as well as the reticence of using GMOs, that both winemakers and researchers have reconsidered the role of non-traditional yeast strains as a path for future development and investigation.

These yeast strains, primarily non-Saccharomyces, are significantly different in their attributes from those used historically in the wine industry. It is only due to increased technological innovation and capability that they are now more available as new tools for winemakers. But why is there this interest in non-Saccharomyces strains and what are the reasons for it? To answer this question we really need to understand what these non-Saccharomyces strains are and what are their attributes.

Non-Saccharomyces Wine Yeast Strains

Non-Saccharomyces yeast is a colloquial term used in the wine industry as a catchall to include many different yeast species. Broadly speaking, these yeast species are present on grapes, on cellar equipment and/or in the cellar environment (through their presence in the air or borne by insects). These include, for example, Torulaspora, Metschnikowia, Pichia, Brettanomyces, Klockera, Lachancea thermotolerans and Candida. This group of yeasts can be broken down into three subsets: aerobic, apiculate and those with a fermentative metabolism. Given the association of apiculate yeasts with the production of volatile acidity (a detriment to wine quality),8 investigation into the utility of these species has been largely confined to the first and third groups. Why would these species be of particular interest?

Recent wine production practices–the use of strong fermenting S. cerevisiae strains, pre- and post-harvest sulfite additions, and better cellar hygiene have minimized the influence of non-Saccharomyces strains that are already in the must and/or cellar. The risks of possible spoilage factors have outweighed any potential positive contributions offered by non-Saccharomyces yeasts.

However, numerous research studies have shown that the benefits of Torulaspora delbrueckii, Lachancea thermotolerans, Pichia kluyveri, Schizosaccharomyces pombe and Metschnikowia pulcherrima can positively impact finished wine quality.11 Rather than providing a general overview of all these strains, we will take a more focused look at one of the aforementioned–Metschnikowia pulcherrima–which is gathering momentum in wine yeast research circles for certain advantageous winemaking properties, namely biocontrol, finished wine quality and possible alcohol reduction.

Metschnikowia pulcherrima has been described as a ubiquitous yeast.5 It can be found on grapes, cherries, flowers, spoiled fruit and on fruit-feeding insects. Increasingly yeast manufacturers and resellers are making Metschnikowia more commercially available.

fig1

Cell morphology of Metschnikowia pulcherrima versus Saccharomyces cerevisiae, with the former (left) having a more elongated, lemon-shape and the latter (right) having a more circular, traditional yeast morphology (40x magnification).

Biocontrol

The common use of sulfur dioxide as an antimicrobial agent in wineries is due to its cost and effectiveness. However, there is a growing trend to reduce sulfur dioxide for wine consumers with allergic reactions to this compound as well as for environmental and stylistic reasons. Because of its ability to produce anti-microbial compounds, Metschnikowia pulcherrima can be used as a natural biological control agent with potential to partially or fully replace sulfur dioxide (depending on the quality of grapes received at the winery).

In particular, Metschnikowia pulcherrima colonies secret pulcherrimic acid (the precursor of pulcherrimin pigment) that depletes iron present in the must making it unavailable to other yeast species, thereby inhibiting their growth.6 This hinders undesired wild spoilage yeast such as Brettanomyces/Dekkera, Hanseniaspora and some Pichia strains (for example Pichia anomala), rather than relying on SO2.

Additionally, it has also been suggested that while iron depletion is a robust inhibitory mechanism, other attributes may also support this antimicrobial aspect such as competition for nutrients. Certain Metschnikowia pulcherrima strains were observed to adversely affect the growth of other microorganisms by consuming nutrients faster than those with which they were competing.5

Furthermore, it may well be that the secretion of enzymes by Metschnikowia pulcherrima such as chitinase and glucanase may also affect the cells of other microorganisms.9 While this certainly requires further investigation it is clear that Metschnikowia pulcherrima does have antimicrobial activity and therefore a potential application in wines where there is a desire to remove sulfur dioxide as an additive on wine labels.

Effects on Wine Flavor/Quality

S. cerevisiae is generally not characterized as being a good producer of exogenous enzymes. Non-Sacharromyces strains, however, have interesting attributes with regards to this. Specifically, Metschnikowia pulcherrima has the following enzymatic activities: ß-glucosidase, protease, glucanase, lichenase, cellulase, xylaniase, sulfite reductase, lipose and ß-lyase.5 A number of these activities are particularly valuable with respect to winemaking. Ricardo Vejarano summarizes some of these benefits:12

Enzyme Benefits

ß-glucosidase
- Release of terpenes from their glycosylated precursors,
- Release of thiols from their cysteinylated precursors.

Protease         
- Improvement of grape must extraction and clarification, wine filtration and stabilization,
- Improvement of foam stability in sparkling wines,
- Increase in the amino acid content, production of aromatic compounds.

ß-lyase 
- Release of thiols from their carbon-sulfur bonded non-aromatic precursors.

It is necessary to recognize that the intensity of the enzymatic activity is not only species- but also strain- dependent.

Aside from these enzymatic effects on wine flavor in recently published work conducted by Varela et al (2021) it was shown that Shiraz and Cabernet Sauvignon wines produced with active dry yeast preparations of Metschnikowia pulcherrima showed increased intensity of some desirable attributes (‘red fruit’ aroma and ‘dark fruit’ flavor) and by low scores for negative descriptors (vegetal aroma and reductive sensory descriptors).11 Their analysis of the volatile composition and subsequent sensory analysis suggests of wines made with Metschnikowia pulcherrima strongly suggest that these yeast preparations can shape both sensory profile and wine style positively.11

Ethanol Reduction

The increase in alcohol levels in wine has seen wines with 14% ABV and above being more commonly produced today. However, due to both health considerations, financial imperatives (taxation) and consumer demands, there has been an increased impetus to lower alcohol levels without being detrimental to wine quality and flavor. One thing common though to all Metschnikowia pulcherrima strains--the fact that they are Crabtree negative–opens up the opportunity to use the species to potentially utilize grape sugars in a non-conventional manner to reduce the ethanol content of wines.

Non-Saccharomyces yeast have shown potential to produce less ethanol in wine2, 10. Largely this is a consequence of the mechanisms for nutrient uptake and those involved in the regulation of the respiro-fermentative metabolism. Generally, these non-Saccharomyces species metabolize sugar without generating ethanol or do so with less efficiency.3

To this end, numerous studies have been conducted seeking to find non-Saccharomyces strains which are able to reduce alcohol levels in wine by taking advantage of this difference in respiratory metabolism. One of the main candidates thus far identified is Metschnikowia pulcherrima. In a study conducted by Angela Contreras et al, Metschnikowia pulcherima used in conjunction with sequential inoculations of S. cerevisiae consistently produced lower ethanol yields in both Chardonnay and Shiraz wines.2 While there was a concomitant rise in glycerol production and some organic acids, these increases were not sufficient to explain fully the ethanol decrease. Unfortunately, the results attained in this study have, to date, not been reproducible in a commercial environment. In part, this was a result of an inability to scale up the trials replicating what was done in the laboratory

In a 2015 study, Pilar Morales et al showed that, in laboratory trials, reductions of up to 3.7% (v) were possible.4 These results were significantly higher than the results of Angela Contreras et al who achieved up to 1.6% reduction.2 In both cases the authors highlighted difficulties in the reproducibility of the results. In the case of Angela Contreras et al this was attributed to the fact that while Metschnikowia pulcherrima potentially reduces ethanol levels it was not, however, a characteristic shared by all the members of the Metschnikowia pulcherrima species.

Difficulties/ Challenges   

Non-Saccharomyces strains have the potential to become valuable tools for winemakers. In several cases, spontaneous fermentations may not have sufficient microflora to gain a big enough foothold to actively out-compete the dominant, resident yeast strain at the start of fermentation. The ability to inoculate with a commercially manufactured, dependable inoculum of non-Saccharomyces strains can potentially be a valuable asset to winemakers both in terms of complexity and other attributes possessed by these strains.

Lachancea thermotolerans, for example, shows a high production of lactic acid able to strongly affect wine pH, sometimes decreasing wine pH by 0.5 units or more during fermentation. In the case of Metschnikowia pulcherrima, this can be in terms of acting as a biocontrol agent or actively contributing positively to the flavor profile of a finished wine or even potentially as a viable method of reducing the alcohol level. Other species likewise carry with them specific attributes which could be useful.

Aside from the low fermentative power of Metschnikowia pulcherrima: it is generally used in conjunction with a sequential inoculation with S. cerevisiae in order to complete fermentation to dryness. Pilar Morales et al noted the difficulties in scaling up from a laboratory scale to commercial ones.4 Further research is needed on strain selection and then subsequently inoculation strategies, the subsequent yeast strain used for sequential inoculation to ensure fermentations go to dryness, and devices and development of specific fermentation nutrients.2

More and more of these advantageous non-Saccharomyces yeasts will be commercially available in the near future as wine yeast researchers get in-depth understanding of their capabilities and yeast manufacturers are able to make them commercially available. No doubt they will become a very important tool for the wine industry moving forward.

 

 

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To know more about non-Saccharomyces yeast, please contact our expert technical advisors:
wineinfo@abbiotek.com
Or visit:
www.maurivin.com/yeast-strains (Next Generation range)
Metschnikowia pulcherrima

 

References

1. Chambers, P. and S. Pretorius. 2010 Fermenting knowledge: The history of winemaking, science and yeast research. EMBO Reports, 11, 914 – 920.

2. Contreras, A. et al. 2014 Evaluation of non-Saccharomyces yeasts for the reduction of alcohol content in wine, Applied Environmental Microbiology, 80, 1670 – 1678.

3. Gonzalez, R. et al. 2013 Yeast respiration of sugars by non-Saccharomyces yeast species: A promising and barely explored approach to lowering alcohol content of wines. Trends in Food Science & Technology, 29, 1, 55-61.

4. Morales, P. et al. 2015 The impact of oxygen on the final alcohol content of wine fermented by a mixed starter culture. Applied Microbiol Biotechnology, 99, 3993–4003.

5. Morata, A. et al. 2019 Applications of Metschnikowia pulcherrima in wine biotechnology, Fermentation, 5, 63.

6. Oro, L. 2014 Antimicrobial activity of Metschnikowia pulcherrima on wine yeasts, J. of Applied Microbiology 116, 1209—1217.

7. Rankine, B. 1968 The importance of yeasts in determining the quality and composition of wines, Vitis 7, 22 - 49.

8. Romano, P. et al., 1992 Higher alcohol and acetic acid production by apiculate wine yeasts, J. of Applied Bacteriology, 73 (2), 126 – 130.

9. Sipiczki, M. 2020 Metschnikowia pulcherrima and Related Pulcherrimin-Producing Yeasts: Fuzzy Species Boundaries and Complex Antimicrobial Antagonism, Microorganisms 8 (7), 1029.

10. Varela, C. et al. 2017 Sensory profile and volatile aroma composition of reduced alcohol Merlot wines fermented with Metschnikowia pulcherrima and Saccharomyces uvarum. Intrnl J. of Food Microbiology, 252, 1 – 9.

11. Varela, C . et al. 2021 Volatile aroma composition and sensory profile of Shiraz and Cabernet Sauvignon wines produced with novel Metschnikowia pulcherrima yeast starter cultures, Australian Journal of Grape and Wine Research 27 (2), 1 - 13

12. Vejarano, R. 2020 Non-Saccharomyces in winemaking: Source of mannoproteins, nitrogen, enzymes and antimicrobial compounds, Fermentation, 6, 76.

 

Published on 01/25/2023
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