Craft beer has gained popularity among consumers in the last decades, and the market share has grown continuously. Sour beer is a well-established category within the craft beer segment. It is characterised by having a pleasant and fresh tartness caused by the organic acids present in the beer. The primary organic acid today is lactate. Various production regimes exist for traditionally produced sour beer, and both spontaneous and inoculated fermentations are utilised.
Traditionally, most lactate in sour beer is produced by lactic acid bacteria before or during alcoholic fermentation. Some organic acids can also derive from acid-producing yeasts. In common for all traditional production regimes is the use of several microorganisms, which makes production challenging to control and relatively laborious. Therefore, a new production regime has been suggested, named “primary souring”, where selected strains of acid-producing non-conventional yeasts are enrolled to undertake both the acid production and alcoholic fermentation, thus simplifying the production.
This thesis mainly aimed to identify non-conventional acid-producing yeasts as potential candidates for the production of sour beer through “primary souring” and testing the candidates in a brewery-like setting. 131 yeast strains were screened for the ability to grow in enzymatically treated brewers’ wort (glucose wort) and reach pH <3.8. 25 strains passed the screening and were selected for further testing in brewers’ wort, and further selection was based on organic acid production, low alcohol production and aroma production. Out of the 25, the yeast Yarrowia lipolytica strain FNC0006 was chosen as the most suitable candidate for further testing as a starter culture for “primary souring” of sour beer. This selection was based on its ability to produce citrate, no production of alcohol and yielding a liquid with a pleasant pineapple-like aroma. Four more Y. lipolytica strains were obtained from the CBS-KNAW collection to test strain differences in fermentation kinetics and citrate production in brewers’ wort containing 30 ppm iso-α-acid. Two strains were selected, the in-house strain FNC0006 and the type strain CBS6124.1 from the CBS-KNAW collection. FNC0006 was selected for its ability to produce a liquid with a pineapple-like aroma and its superior growth characteristic. CBS6124.1 was selected due to its superior ability to lower pH and synthesise citrate and iso-citrate.
Y. lipolytica is a dimorphic yeast, which is generally regarded as safe (GRAS), Crabtree-negative and strictly aerobic. It is extensively researched in biotech as a producer of multiple high-value metabolites, e.g., intermediates from the tricarboxylic acid cycle; citrate, iso-citrate, succinate, and α-ketoglutarate as well as aroma compounds (2-phenylethanol and γ-decalactone). As a starter culture, very few documented uses are found, and even fewer in a beverage context, despite potential as a producer of functional and organoleptic pleasing beverages.
Y. lipolytica FNC0006 was tested for aroma production and fermentation kinetics in a setting that would be possible to create in a brewery setting, which means fermentation in brewers’ wort containing iso-α-acid, at high and low aeration levels and at 20 ℃ and 30 ℃. It was found that FNC0006 could grow under the given conditions; it was, however, inhibited. It only consumed glucose and fructose, and despite meagre sugar consumption, it was able to assimilate 75-80% of the total amino acid in the high aeration treatments and synthesise 2-phenylethanol.
Y. lipolytica CBS6124.1 and FNC0006 were subsequently tested for fermentation kinetics and organic acid production in glucose wort at aerobic conditions (1.0 vvm). The effect of iron addition in the wort on fermentation kinetics and organic acid production was evaluated.
Significant differences between the two strains of Y. lipolytica were found. FNC0006 produced much more citrate and iso-citrate than CBS6124.1 and had a faster general metabolism under the given conditions. No effect of interest of the iron addition was found for the FNC0006 strain.
Metabolism was, however, faster in the fermentation with iron added for CBS6124.1.
It is concluded that sufficient aeration of the medium is vital, and using glucose wort as medium is the most promising for producing sour beer when using Y. lipolytica as a starter. Besides this Y. lipolytica have multiple traits of interest for producing sour beer through “primary souring”.
However, some questions do remain unanswered before a conclusion can be made. Do the metabolites produced by Y. lipolytica actually contribute to the wanted traits for a sour beer, what is the scalability of the process, and how is it technically possible to reach a proper aeration level in a traditional brewery?