New and improved yeast and bacterial starter cultures-novel attributes, process efficiency and wine distinctiveness
Abstract
This project continued our research to develop an improved microbial toolkit of yeast and bacteria strains with desirable fermentation traits and wine aroma attributes. Methods investigated included directed evolution and CRISPR Cas9 gene editing tools. A Systems Biology approach was used, to provide a sound understanding of the genetic basis of improved characteristics. The key outputs were a diverse collection of microbial strains, mainly comprising non-Saccharomyces yeast species and lactic acid bacteria. These were isolated from the environment, industry and scientific collections. We have also advanced the understanding of what are considered as new risks to winemaking i.e. the use of juice dilution and the presence of bacteriophage. We found that juice dilution reduced fermentation duration by up to 28% when used within legal limits, and also results in many changes to the volatile profile of finished wines, which are not fully repaired through nutritional supplementation. Bacteriophage were found in almost all samples analysed and we have preliminary evidence suggesting that under certain conditions, purified phage can delay malic acid utilisation by lactic acid bacteria.
The outcomes of this project have the potential to generate many benefits for winemakers, primarily by decreasing the risk of problem fermentations.
The key Outputs of this project were a diverse collection of microbial strains, mainly comprising non-Saccharomyces species (yeast) and lactic acid bacteria. These were isolated from the environment, industry and scientific collections. The yeast and some of the bacterial strains were classified to species level and then screened for traits considered to be desirable to the wine industry. Where required, methodology for these screens was designed and improved. We analysed strains for fermentation reliability, including (for bacteria) malolactic fermentation in the presence of high SO2, the enzymatic activities of protease, lipase, β-glucosidase as well as the ability to utilise the spoilage compound acetic acid and also for antimicrobial activity against the spoilage organism Brettanomyces bruxellensis. Strains with these capabilities were identified in each of these categories. The most promising strains were analysed in small industrial scale fermentations where finished wines were then examined by sensory panels and for volatile chemical profiles. Here the highlights included four non-Saccharomyces strains from the species Debaryomyces hansenii, Aureobasidium pullulans, Kazachstania servazzii and Kazachstania aerobia, each contributing interesting floral profiles to wine aroma, as well as one Saccharomyces cerevisiae (C7H_B4) improved for fermentation reliability using directed evolution and found to also contribute to desirable wine aroma. We also sequenced the genomes of six strains evolved through directed evolution and the two Kazachstania sp. of interest and isolated from the environment. Comparison of genetic differences of these to our Fermentation Relevant Yeast Gene Database (also expanded in this project) advanced knowledge of what the causal genetic modifications may be for certain phenotypes of interest in a wine context. We then tested some of these hypotheses and advanced the methodology of CRISPR Cas9 gene editing in wine yeast through construction and analysis of the performance of six proof-of-concept engineered strains in the most commonly used commercial wine yeast strain in Australia (Lalvin EC1118®). Most helpfully, this also included using a technique that under current Australian legislation would see the modified strain being classified as non-GMO.
We have also advanced the understanding of what are considered as new risks to winemaking; the use of juice dilution and the presence of bacteriophage. Here we found that juice dilution does indeed reduce fermentation duration, by up to 28% when used within legal limits, and also results in many changes to the volatile profile of finished wines, often not repaired through nutritional supplementation. Bacteriophage were found in almost all samples analysed and we have preliminary evidence suggesting that under certain conditions, purified phage can delay malic acid utilisation by lactic acid bacteria. Since this project has included many lines of research, there have also been a number of other novel findings not originally proposed as Outputs. Wherever possible and appropriate, we have attempted to capture any such findings. One example is that as part of attempting to understand the mechanisms behind SO2 tolerance of lactic acid bacteria, we also discovered that one of the key influences is the availability of methionine during alcoholic fermentation. A lack of this amino acid results in elevated SO2 production by yeast, which in turn, inhibits malolactic fermentation by lactic acid bacteria.
Through this project we have managed to deliver many advances to the field of wine microbiology and our findings have been disseminated in many formal publications and presentations:
- 14 scientific journal articles (2 more in draft form)
- Two technical articles
- One technical brochure
- 13 conference presentations
- Seven accepted theses (with a further two PhD students completing shortly)
- Lectures to undergraduate and postgraduate Oenology/Viticulture and Biotechnology students
Summary
The use of yeast and bacteria, specifically selected for desirable traits leading to reliable and predictable primary and secondary fermentation remains the most common method to ensure beneficial fermentation outcomes in new-world industrial winemaking. Even though many commercial strains are available in the market, feedback from the Wine Australia Research Advisory Committee, as well as surveyed winemakers and suppliers during this project, is that issues with fermentations still exist that are directly related to microbial failure. This is also exacerbated by changes in the technical requirements of microbes, such as being able to perform even when the composition of grape musts may vary due to changes in viticultural practices and climate change. The core objective of this project was to improve the wine microbial toolkit available by providing strains and information regarding methods for improvement and use. This included extending our previous research on the use of directed evolution for strain improvement as well as investigating potential new or untested microbial issues such as the use of juice dilution and the impact of bacteriophage. We specifically targeted industry requirements and our approach was holistic, whereby the techniques used to isolate and/or improve novel microbes incorporated a Systems Biology approach to provide a sound understanding of the genetic basis of improved characteristics. It further allowed us to mine for other genetic characteristics that may be of use for future strain improvement. We expect that reliable fermentation outcomes will translate into further improvements to sustainable and economical wine production with enhanced or consistent wine quality.