Co-cultivation of non-conventional yeast with Saccharomyces cerevisiae to increase the aroma complexity of fermented beverages

<p>Yeast are used as workhorses to convert hopped wort into beer. Conventionally, such yeasts belong to the genus <em>Saccharomyces</em> and most research on fermentation of wort for the production of beer has focussed on the species <em>Saccharomyces</em> cerevisiae and <em>Saccharomyces pastorianus</em>. Recently, there is an increasing interest in unravelling features of non-conventional yeast species for beer innovation. In this thesis, features of yeast isolates belonging to the species: <em>Cyberlindnera fabianii</em>, <em>Pichia kudriavzevii</em> and <em>S. cerevisiae</em> (all isolated from fermented masau (<em>Ziziphus mauritiana</em>) fruits in Zimbabwe), were studied with focus on aroma production. Additionally, a novel approach was used to apply these yeasts in co-cultivation with Brewers’ yeast (<em>S. cerevisiae</em>) for beer innovation.</p> <p>The characteristics and quality of the beer are mainly determined by aroma compounds in the final product such as esters, alcohols, aldehydes and acids. Yeast use various metabolic pathways such as glycolysis, the fermentative pathway, the tricarboxylic acid (TCA) cycle and the Ehrlich pathway to produce aroma compounds or the precursors for the synthesis thereof (Chapter 1). Among the aroma compounds, esters are of major importance, especially since they are perceived by the human olfactory system at very low concentrations. In general, esters are desirable compounds in beers due to their fruity flavour. Examples are isoamyl acetate (banana), isobutyl acetate (fruity, sweet), phenylethyl acetate (rose, apple, honey), ethyl acetate (sweet pear), ethyl hexanoate (apple, aniseed) and ethyl octanoate (sour apple). Together with an extensive range of other volatile organic compounds (VOCs) these compounds were previously profiled using headspace solid-phase-micro-extraction gas-chromatography mass-spectrometry (GCMS). Interestingly, comparative profiling of aromas showed that <em>C. fabianii</em> produces significantly higher amounts of isoamyl acetate and ethyl acetate compared to <em>S. cerevisiae</em>. </p> <p>It has been suggested in literature that products of the Ehrlich pathway, so called “fusel alcohols”, can function as signalling molecules for invasive growth upon nitrogen limitation. This suggested that nutrient limitation could affect growth performance and production of aroma compounds. Therefore, in Chapter 2, the metabolic and morphological response of <em>S. cerevisiae</em>, <em>C. fabianii</em> and <em>P. kudriavzevii</em> was analysed upon nitrogen and/or glucose limitation on semi-solid (agar) media. All three yeasts showed a change in growth mode upon nitrogen and/or glucose limitation. Scanning electron microscopy was used to unravel the cell organisation of <em>C. fabianii</em> and <em>P. kudriavzevii</em> grown under nitrogen limitation. This revealed the power of cell-cell adhesion for penetrative growth and the formation of meta-filaments or pseudo-hyphae to extend the cell surface area. Such changes in growth mode may be of relevance for solid-state fermentation processes, such as fruit fermentations, by enhanced substrate penetration of yeast. Notably, a significant increase in the production of esters (ethyl acetate, ethyl propanoate, isobutyl acetate and isopentyl acetate) by <em>C. fabianii </em>and <em>P. kudriavzevii</em> was found under conditions of nitrogen limitation. Understanding the relationship between nitrogen limitation and ester formation gives more insight into the ability to steer ester formation by nutrient availability in wort fermentations.</p> <p>The amount and type of esters are important determinants of the final flavour characteristics of the beer. Therefore, the diversity in ester production between the three yeast species was investigated by studying enzymatic reactions involved in the production (synthesis) of esters and their degradation (hydrolysis) and to link this to volatile organic compound profiles (Chapter 3). The amount and type of esters depends on substrate availability and a combination of enzyme activities supporting the synthesis and hydrolysis of the different esters formed. Esters are generally formed by a condensation reaction of an alcohol with ac(et)yl CoA by a so-called alcohol acetyltranferase (AATse). The products formed can generally be subdivided into two groups; acetate esters and medium chain fatty acid (MCFA) esters. Alcohols are formed by reduction of aldehydes by alcohol dehydrogenases (ADH) using either the fermentative or Ehrlich pathway. Comparative analysis of the specific ADHs and acetate ester hydrolysing activity and subsequently linking these data with the distinct aroma profiles of the three yeasts revealed that the acetate ester hydrolysing activity is a key step in determining the final pool size of acetate esters found in the fermentation broths (Chapter 3). Under the experimental conditions, <em>C. fabianii</em> showed the lowest acetate ester hydrolysing activity correlating with higher extracellular levels of acetate esters indicating the suitability of this yeast for use in co-cultures with brewers’ yeast with the objective to enable the enrichment of acetate esters in the fermentation process (Chapters 4 and 5).</p> <p>Nowadays, there is large interest of consumers in specialty beers such as beers low in alcohol (health awareness) and/or richer is fruity flavours (specialty beers), and this has significantly stimulated the quest for new methods, practices and yeast strains to produce such beers. In chapter 4 an innovative approach is described using co-cultivations of brewers’ yeast and <em>C. fabianii</em> to steer wort fermentation performance. Various ratios of brewers’ yeast over <em>C. fabianii </em>were inoculated in wort. A dose response relationship was observed, where a higher initial dose of <em>C. fabianii</em> leads to lower alcohol production and a more complex aroma bouquet. Interestingly, specific esters, i.e. ethyl acetate (sweet pear), ethyl octanoate (sour apple), ethyl decanoate (waxy, sweet apple), ethyl 9-decenoate (fruity, fatty) and ethyl dodecanoate (fruity, waxy), were found in higher levels in co-cultivation compared to both mono-cultivations indicating metabolic interactions. The reduced ethanol production in the co-culture could be explained by inhibition of Brewers’ yeast performance by <em>C. fabianii </em>in the co-cultivations. Further investigations revealed that this growth inhibition is caused by competition for oxygen between brewers’ yeast and <em>C. fabianii</em>. Depletion of oxygen caused inhibition of growth of brewers’ yeast since it needs oxygen to synthesize ergosterol that is required for cell membrane synthesis under anaerobic conditions (Chapter 4).</p> <p>The interaction between brewers’ yeast and <em>C. fabianii</em> in co-cultivation can be described using dynamic modelling (Chapter 5). A dynamic model was developed based on brewers’ yeast and <em>C. fabianii</em> in mono-cultivation and fitted to experimental data. The two models were combined and the same parameter settings were used to predict the fermentation outcome of brewers’ yeast and <em>C. fabianii</em> in co-cultivation. The model was experimentally validated using inoculation ratios of 1:10 and 1:100 brewers’ yeast over <em>C. fabianii</em>. Additionally, the use of dynamic modelling supported the hypothesis that competition for oxygen between brewers’ yeast and <em>C. fabianii</em> results in inhibition of brewers’ yeast fermentation performance. Interestingly, prediction of aroma formation in co-cultivation, especially that of specific esters, appeared to be more challenging due to metabolic interactions resulting in MCFA-esters contributing to fruity aromas, and this aspect requires further study.</p> <p>The results and findings obtained in the experimental chapters (Chapter 2-5) are further discussed in Chapter 6. Unravelling features of non-conventional yeast generates novel opportunities for beer innovation. Application of <em>C. fabianii</em> in co-cultivation with brewers’ yeast in wort fermentation offers a novel approach in product innovation resulting in low alcohol beers with enriched aroma bouquet . Finally, the developed dynamic model may be used to predict fermentation outcomes of brewers’ yeast with other non-conventional yeast species.