Metabolomics aims to identify the changes in endogenous metabolites of biological systems in response to intrinsic and extrinsic factors. This is accomplished through untargeted, semi-targeted and targeted based approaches. Untargeted and semi-targeted methods are typically applied in hypothesis-generating investigations (aimed at measuring as many metabolites as possible), while targeted approaches analyze a relatively smaller subset of biochemically important and relevant metabolites. Regardless of approach, it is well recognized amongst the metabolomics community that gas chromatography-mass spectrometry (GC–MS) is one of the most efficient, reproducible and well used analytical platforms for metabolomics research. This is due to the robust, reproducible and selective nature of the technique, as well as the large number of well-established libraries of both commercial and ‘in house’ metabolite databases available. This review provides an overview of developments in GC–MS based metabolomics applications, with a focus on sample preparation and preservation techniques. A number of chemical derivatization (in-time, in-liner, offline and microwave assisted) techniques are also discussed. Electron impact ionization and a summary of alternate mass analyzers are highlighted, along with a number of recently reported new GC columns suited for metabolomics. Lastly, multidimensional GC–MS and its application in environmental and biomedical research is presented, along with the importance of bioinformatics. The purpose of this review is to both highlight and provide an update on GC–MS analytical techniques that are common in metabolomics studies. Specific emphasis is given to the key steps within the GC–MS workflow that those new to this field need to be aware of and the common pitfalls that should be looked out for when starting in this area.

Review of recent developments in GC-MS approaches to metabolomics-based research.

It is well established that non-Saccharomyces wine yeasts, considered in the past as undesired or spoilage yeasts, can enhance the analytical composition and aroma profile of the wine. The contribution of non-Saccharomyces yeasts, including the ability to secret enzymes and produce secondary metabolites, glycerol and ethanol, release of mannoproteins or contributions to color stability, is species- and strain-specific, pointing out the key importance of a clever strain selection. The use of mixed starters of selected non-Saccharomyces yeasts with strains of Saccharomyces cerevisiae represents an alternative to both spontaneous and inoculated wine fermentations, taking advantage of the potential positive role that non-Saccharomyces wine yeast species play in the organoleptic characteristics of wine. In this context mixed starters can meet the growing demand for new and improved wine yeast strains adapted to different types and styles of wine. With the aim of presenting old and new evidences on the potential of non-Saccharomyces yeasts to address this market trend, we mainly review the studies focused on non-Saccharomyces strain selection and design of mixed starters directed to improve primary and secondary aroma of wines. The ability of non-Saccharomyces wine yeasts to produce enzymes and metabolites of oenological relevance is also discussed.

/pdf/past-and-future-of-non-saccharomyces-yeasts-from-spoilage-17ewdb5inh.pdf

Past and Future of Non-Saccharomyces Yeasts: From Spoilage Microorganisms to Biotechnological Tools for Improving Wine Aroma Complexity

Commercial Saccharomyces strains are usually inoculated to ferment alcoholic beverages due to their ability to convert all fermentable sugars into ethanol. However, modern trends in winemaking have turned toward less known, non-Saccharomyces yeast species. These species perform the first stages of natural spontaneous fermentation and play important roles in wine variety. New alcoholic fermentation trends have begun to consider objectives other than alcohol production to improve flavor diversity. This review explores the influence of the most used and commercialized non-Saccharomyces yeast, Torulaspora delbrueckii, on fermentation quality parameters, such as ethanol, glycerol, volatile acidity, volatile profile, succinic acid, mannoproteins, polysaccharides, color, anthocyanins, amino acids, and sensory perception.

The impact of Torulaspora delbrueckii yeast in winemaking

Contribution of non-Saccharomyces yeasts to wine volatile and sensory diversity: A study on Lachancea thermotolerans, Metschnikowia spp. and Starmerella bacillaris strains isolated in Italy.

Starter cultures are defined as selected microbial preparations used to increase the efficiency of fermentation processes. In the food industry, numerous microbial cultures are used to ensure the p...

pdf/a-review-of-selection-criteria-for-starter-culture-i6jvkk8p3d.pdf

A Review of Selection Criteria for Starter Culture Development in the Food Fermentation Industry

Saccharomyces cerevisiae (SC) is the main driver of alcoholic fermentation, however for aroma and flavor formation in wine, non-Saccharomyces species can have a powerful effect. This study aimed to compare untargeted volatile compound profiles from SPME–GCxGC-TOF-MS and sensory analysis data of Sauvignon blanc wine inoculated with six different non-Saccharomyces yeasts followed by SC. Torulaspora delbrueckii (TD), Lachancea thermotolerans (LT), Pichia kluyveri (PK) and Metschnikowia pulcherrima (MP) where commercial starter strains, while Candida zemplinina (CZ) and Kazachstania aerobia (KA), were isolated from wine grape environments. Each wine showed a distinct profile both sensorially and chemically. SC and CZ wines were the most distinct in both of these cases. SC wine had guava, grapefruit, banana, and pineapple aromas while CZ wine was driven by fermented apple, dried peach/apricot, and stewed fruit as well as sour flavor. Chemically over 300 unique features were identified as significantly different across the fermentations. SC wine had the highest number of esters in the highest relative concentration but all the yeasts had distinct ester profiles. CZ wine displayed the highest number of terpenes in high concentration but also produced a large amount of acetic acid. KA wine was high in ethyl acetate. TD wine had fewer esters but three distinctly higher thiol compounds. LT wine showed a relatively high number of increased acetate esters and certain terpenes. PK wine had some off odor compounds while the MP wine had high levels of methyl butyl-, methyl propyl-, and phenethyl esters. Overall, this study gives a more detailed profile of these yeasts contribution to Sauvignon blanc wine than previously reported.

Untangling the wine metabolome by combining untargeted SPME–GCxGC-TOF-MS and sensory analysis to profile Sauvignon blanc co-fermented with seven different yeasts

/pdf/metabolomic-profiling-of-non-saccharomyces-yeasts-in-wine-5bnto6zz43.pdf

Metabolomic profiling of non-Saccharomyces yeasts in wine

Background and Aims
This study evaluated the impact that the non-Saccharomyces yeasts, Torulaspora delbrueckii (TD), Lachancea thermotolerans (LT), Pichia kluyveri (PK), Metschnikowia pulcherrima (MP), Candida zemplinina (CZ) and Kazachstania aerobia (KA), in sequential inoculation with Saccharomyces cerevisiae (SC), had on the volatile chemical profile of Shiraz wine.

Methods and Results
Shiraz grape must was inoculated with monocultures of the non-Saccharomyces yeasts, which were allowed to ferment until 2% v/v ethanol concentration was reached at which point, SC was added to complete the alcoholic fermentation. The control was SC in monoculture. The final wines were subjected to solid phase microextraction-GC × GC-time of flight-MS to evaluate the untargeted volatile metabolite profile of each treatment. Each fermentation produced a unique chemical profile. The LT–SC sequential fermentation was the most significantly different from the control primarily in the ester, alcohol and terpene profile. The KA–SC sequential fermentation had the highest amount of volatile acidity, and the PK–SC sequential fermentation had a relatively high amount of acetaldehyde and a few esters. The MP–SC sequential fermentation also revealed a higher concentration of several esters. The TD–SC sequential fermentation was notable for its lack of a distinct pattern in comparison with that of the other fermentations.

Conclusions
Given these characteristics, the LT–SC sequential fermentation showed the most potential for increased chemical complexity of the Shiraz volatile profile.

Significance of the Study
The results demonstrate that there is no clear, singular trend for how different, non-Saccharomyces species of yeast – once thought to be wholly unfavourable in wine fermentations – will impact wine chemistry, flavour or quality. Each species presents unique metabolic characteristics, many of which could be beneficial, and their potential application in the wine industry should be considered.

Effect of non-Saccharomyces yeasts on the volatile chemical profile of Shiraz wine

Saccharomyces cerevisiae is the main driver of alcoholic fermentation. It is typically inoculated at high levels to ensure successful implantation as well as reduce the risks of stuck fermentations and off-flavor production. However, winemakers have found that wines produced with only S. cerevisiae can be lacking in complexity compared to fermentations where non-Saccharomyces yeasts are more active. This study sought to understand the early fermentation characteristics of Kazachstania gamospora, Lachancea thermotolerans, Metschnikowia pulcherrima, Torulaspora delbrueckii and Zygosaccharomyces kombuchaensis in both Sauvignon blanc and Syrah musts. S. cerevisiae was used as a control. Solid-phase microextraction coupled to GC–MS was used to evaluate the musts once they reached 2% ethanol concentration. The method targeted 90 different compounds known to occur in wine and/or be produced by yeast during fermentation. For the first time, K. gamospora and Z. kombuchaensis have been studied in the context of wine. While the other yeasts are commercially available starter cultures, they have never been profiled this extensively. Analysis showed that each yeast profile was unique and different based on the must. The non-Saccharomyces yeasts produced lower concentrations of esters, alcohols and terpenes with the exception of K. gamospora which produced more total esters than the control.

Early fermentation volatile metabolite profile of non-Saccharomyces yeasts in red and white grape must: A targeted approach

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Untangling the wine metabolome by combining untargeted SPME–GCxGC-TOF-MS and sensory analysis to profile Sauvignon blanc co-fermented with seven different yeasts

Metabolomic profiling of non-Saccharomyces yeasts in wine

Effect of non-Saccharomyces yeasts on the volatile chemical profile of Shiraz wine

Early fermentation volatile metabolite profile of non-Saccharomyces yeasts in red and white grape must: A targeted approach