Brewing Efficacy of Non-Conventional Saccharomyces Non-Cerevisiae Yeasts

Consumer demands for new sensory experiences have driven the research of unconventional yeasts in beer. While much research exists on the use of various common Saccharomyces cerevisiae strains as well as non-Saccharomyces yeasts, there exists a gap in knowledge regarding other non-cerevisiae Saccharomyces species in the fermentation of beer, outside that of S. pastorianus. Here, five distinct species of Saccharomyces from the UC Davis Phaff Yeast Culture Collection, as well as one interspecies hybrid from Fermentis, were chosen to ferment 40 L pilot scale beers. S. kudriavzevii, S. mikatae, S. paradoxus, S. bayanus, and S. uvarum yeasts were fermented in duplicate, with one fermenter in each pair receiving 10 g/L dryhop during fermentation. Analytical measurements were made each day of fermentation and compared to controls of SafAle US-05 and SafLager W 34/70 for commercial brewing parameters of interest. Finished beers were also analyzed for aroma, taste, and mouthfeel to determine the flavor of each yeast as it pertains to brewing potential. All beers exhibited spicy characteristics, likely from the presence of phenols; dry-hopping increased fruit notes while also increasing perceived bitterness and astringency. All of the species in this study displayed great brewing potential, and might be an ideal addition to beer depending on a brewery’s desire to experiment with flavor and willingness to bring a new yeast into their production environment.

In addition to novel yeast-derived flavors, brewers are increasingly turning to dryhopping to enhance their consumers' sensory experience. Historically this procedure of adding hops (Humulus lupulus) cones to beer when fermentation is active or finished was performed to provide packaging and transport stability [27,28]. Relatively more recently with the rise of Craft Brewers, dry-hopping with pellets or advanced hop products [29] has become a common tactic used by brewers desiring to add interesting flavors and aromas to their beer [30].
All Saccharomyces yeast species that have been found to produce ethanol from carbohydrate sugar sources have been classified as part of the Saccharomyces sensu stricto (Sss) complex [31][32][33]. While the Sss currently contains ten distinct species, only eight have been linked to alcoholic beverage fermentation (Fig. 1). Use of S. cerevisiae and S. pastorianus have long been known for their use in alcoholic beverage production, but the Sss contains several nonconventional species. S. kudriavzevii, S. mikatae, S. paradoxus, S. bayanus, and S. uvarum that have already shown potential for alcoholic beverages, and have been identified in fermentations of wine, tepache, cider, chicha, palm wine, umqombothi, and other beverages [19,[34][35][36][37][38][39]. Many of these fermented beverages, however, contain mixed cultures of yeasts and sometimes bacteria, in addition to naturally formed interspecies hybrids between two or more different Saccharomyces species [24,40]. To date, none of these species have been evaluated in monoculture fermentations in a beer brewing context, but their efficacy has been previously reviewed [41]. parenthesis to indicate it was derived from multiple hybridization events [42]. S. pastorianus is shown as a genetic hybrid of S. eubayanus and S. cerevisiae [21]. Use in fermented beverages is indicated with plus signs (+) for current commercial use, with S. cerevisiae and S. pastorianus exhibiting the most ubiquitous use in beer, and negative signs (−) for no known use. S. cariocanus is known to be harboring just four translocated chromosomes different than S. paradoxus [43]. S. jurei has very recently been proven to have brewing potential [44].
First isolated from oak trees of western Europe, S. kudriavzevii is a wild-type yeast that has been sequenced to contribute 23-96% of its genome to hybrids with S. cerevisiae [16,19,45].
While no commercial examples of its use in beer fermentation exist, S. kudriavzevii has been isolated from mixed-cultures of farmhouse ciders in France and draft beer systems in Germany to New Zealand [46,47]. Due to its propensity to hybridize, this yeast has even been found as part of the genetic makeup in Belgian Trappist ale strains from Chimay, Westmalle, and Orval [48].
S. kudriavzevii is a cryophilic species and is currently used to ferment wines at lower temperatures (10 °C to15 °C) in Europe and Australia [19,49]. Because it thrives at low temperatures and may have aromas similar to Belgian beers, S. kudriavzevii has potential for use in the production of hoppy lager beers in the brewing industry.
S. paradoxus has been found in African umqombothi [38] and white wine fermentations previously [50], but has only been studied for its beer brewing potential (at 15 °C) very recently, since the inception of this research [22]. S. paradoxus was one of the first species isolated as a member of the Sss outside of S. pastorianus and S. cerevisiae and is typically found in tree sap of Northeastern Europe [51]. Being a wild-type yeast species suggests S. paradoxus may produce interesting volatile aroma compounds at warmer (18 °C to 24 °C) ale temperatures [52].
Saccharomyces mikatae is a wild yeast that contributes to genetic hybrids from interspecies hybridization events with S. cerevisiae and S. paradoxus [53], and was first isolated from soil and decaying leaves in Japan [43]. S. mikatae was shown to form a biofilm on the surface of liquid media (pellicle) after twenty-five days at 20 °C, similar to wild-type strains [43]. It produced fruity, banana, floral, and sweet perfume aromas in white wine, and ferment slowly, perhaps all due to its diversion from the S. cerevisiae parent genome [54,55]. Both S. paradoxus and S. mikatae offer unique characteristics that might be of interest to craft brewers creating beer at ale fermentation temperatures.
Saccharomyces bayanus was previously thought to be the parent of the lager strain, S.
pastorianus [21,47,56], but the hybridization event that produced lager brewing yeast is now proven to have occurred between S. cerevisiae and S. eubayanus [25,34,57,58]. S. bayanus has been characterized as its own species within the Sss, but in order delineate it from S. eubayanus and S. uvarum, it is commonly referred to as S. bayanus var. bayanus [21,42]. Genetic analysis of organisms in beer fermentations have identified S. bayanus as part of blended cultures due to its chromosomal similarity to S. pastorianus [26], but it is most common as a solitary species in wine fermentations [58]. A close relative, Saccharomyces uvarum, was once was thought to be a variant of S. bayanus, but has since been confirmed as a distinct species [59]. S. uvarum has been found to be part of the mixed culture of spontaneously fermented wines [36], as well as an interspecies hybrid known in some Norwegian kveik strains [17]. Both S. bayanus and S. uvarum exhibit increased levels of isoamyl acetate in wine and brandy [60,61], and might contribute similar flavor to beer.
This interspecies hybrid has been created through a LeSaffre R&D program to select a yeast strain that exhibits increased expression of aromatic terpenes. New research has identified these terpene compounds as some of the most impactful on dry-hopped beer aroma [30,63] through biotransformation with glycosides and alcohols to produce unique aroma characteristics [64].
While this yeast was developed for wine fermentations, it may be of great interest to brewers making dry-hopped beers, and was therefore selected for this study.
While there is much research regarding the use of some of these species in a laboratory scale or wine fermentation, work remains for their efficacy and commercial use in the production of beer. Additionally, little to no sensorial analysis exists on the use of any of these Saccharomyces spp. in the fermentation of beer, most notably at ale fermentation temperatures (18-20ºC) or in dry-hopped beers. The aim of this study is to assess the brewing potential of the non-conventional non-cerevisiae Saccharomyces species outlined above by assessing fermentation kinetics and performance, yeast abundance and viability post-fermentation for serial re-pitching, as well as the flavor characteristics of the resultant beer. Beers in this study will be run as both dry-hopped and standard fermentations due to the pervasiveness of dryhopping in the American craft brewing industry. While the most widely used non-cerevisiae Saccharomyces species is S. pastorianus, it will not be discussed here as much research already exists on its brewing potential.

a. Experimental Beers
A total of eight all-malt pilot scale brews were performed on the 1.8 hL Anheuser-Busch Research Pilot Brewery at the University of California, Davis. Brewing parameters, as well as the malt, hops, water chemistry, mashing regime, pH, boiling parameters, and knockout temperatures followed the same method as outlined in previous research [65]. The experimental beer recipe was similar to an American Pale Ale or Session IPA, with a target original gravity of 10 ºP, to yield a 4.2% (v/v) alcohol beer under standard ale fermentation conditions. Wort from each of the eight brews was split evenly by volume between four 56 L fermenters, to fill each with approximately 40 L of cooled wort.

b. Yeasts
Saccharomyces yeasts sourced from the University of California, Davis, Phaff Yeast Culture Collection (phaffcollection.ucdavis.edu) included the type strains of S. kudriavzevii, S. mikatae, S. paradoxus, S. bayanus, and S. uvarum. Additionally, the control S. cerevisiae and S. pastorianus species and one S. cerevisiae x S. bayanus hybrid were provided by Fermentis (Table 1). Yeasts from the Phaff Collection were revived from cryogenic storage and streaked onto potato dextrose agar (PDA) plates and incubated for 2 days at 30 ºC before being moved to room temperature storage until propagation. Yeasts from Fermentis were provided as an active dry yeast with the emulsifier E491 (sorbitan monostearate) and stored at 4 ºC until propagation. propagation wort and propagated stepwise over the course of 11 days following the methods outlined in previous research [65] and  Each unique Saccharomyces species ( or "terminal gravity" [70] was defined here as a change of less than 0.10 ºP gravity for two simultaneous days following dry-hop.
After fermentation was completed, all beer in all fermenters except the S. cerevisiae and S.
pastorianus controls were cold conditioned at 0.0 ºC for two days to allow for natural clarification. Yeast and hops were removed from the bottom of the cylindroconical fermenter before the beer was transferred to a 19.6 L Sankey keg for carbonation. All were packaged from the kegs into CO2-purged 0.95 L (32 oz.) "Crowler" cans (Ball Corporation; Westminster, CO, USA) and stored below 4.0 ºC until sensory analysis and shipping.

d. Sample Collection and Preparation
Fermenting beers were aseptically sampled daily within a two-hour window of the time of knockout transfer of wort to fermenter. 50 mL conical tubes of each sample were centrifuged (ThermoFisher Scientific; Waltham, MA, USA) at 20 °C and 3000 x g RCF for five minutes.
The clarified supernatant was then degassed for five minutes using the degas setting on a VWR

e. Sensory Analysis
Each set of packaged beer from an individual fermentation was assigned a randomly selected three-digit code in order to ensure blind analysis of experimental samples. The willing members of the UC Davis Brewing and Malting Science laboratory team (n = 7) used a modified consensus method [72] with check-all-that-apply (CATA) [73] in two tastings to choose appropriate aroma descriptors from the DraughtLab Beer Flavor Map © (Fig. 3)  The common aroma descriptors were parsed down to the twelve most recurrent amongst the experimental beers. Each of these twelve descriptors, the five accepted taste modalities, and three recurrent mouthfeel descriptors from the consensus panel were placed on a 9-point intensity scale for scoring by the local brewery panelists ( Table 2). and rated each on a 9-point intensity scale from "none" to "extremely strong" [74,75]. Training, methods and frequency of sensory panels varied from brewery to brewery, however it was minimally required that the panelists were able to accurately distinguish dry-hopped from nonhopped beer and identify German, Belgian, and American ale strain characteristics. The total sample group to perform sensory analysis on the experimental beers consisted of 51 panelists (36 male and 15 female), ranging in age from 24 to 61. No panelists had medical reasons for not consuming alcohol. Score each attribute by circling a number, with 0 = none to 9 = extremely strong Aroma: Cereal: Grainy, Biscuit, Cracker, Wort

a. Pilot Fermentations
Ten total brews were performed for the twenty fermentations, with a mean original gravity (O.G.) of 10.2º Plato (±0.36), and a higher brewhouse efficiency than expected for the recipe designed at 10.0º P (Fig. 4). Fermentations were carried out at 20.0ºC, standard ale temperatures, and analytical parameters were measured on each day of fermentation. Results were compared with the two control strains, S. cerevisiae US-05 and S. pastorianus W 34/70. Vigorous fermentations of the control species suggest an adequate yeast pitching rate, nutrients levels, and wort aeration were utilized. All fermentations reached terminal gravity within two weeks, with the exception of S. uvarum UCDFST 11-512, which took fifteen days for the non-hopped fermentation but only eleven days for the dry-hopped fermentation ( Table 3). However, all average fermentation lengths were not shown to be statistically different between dry-hopped and non-hopped fermentations (p > 0.05). These fermentation lengths indicate all the yeasts studied here are viable candidates for production breweries that normally ferment lagers, but perhaps too long for breweries that normally produce ales. Conditioning time was not accounted for in this study, as all fermentations were deemed terminal based on gravity instead of from the presence of secondary metabolites, such as diacetyl or acetaldehyde concentrations.  hopped beers on day of terminal gravity, stained with methylene blue, as per standard procedure (69). Viability was not performed on dry-hopped beers due to interference from hops in suspension. Fermentation length as given in days to achieve final gravity. Strain listed as "Hybrid" is interspecies hybrid of S. bayanus x S. cerevisiae from LeSaffre R&D. 10.0 ± 1.0 8 * indicates the mean of the three biological replicates **data not recorded All yeasts measured for viability showed greater than 80.0 % living cells at the end of fermentation, signifying a potential for serial re-pitching in a commercial setting. Viability was not measured on the two control strains, US-05 and W 34/70, as their ability for propagation and serial re-pitching has been extensively studied [76][77][78]. Viability data for S. bayanus x S. cerevisiae HD T18 was not available and should be further evaluated as it is not standard practice to re-pitch wine yeasts due to ethanol toxicity [79].
When comparing the dry-hopped and non-hopped fermentations, average differences for alcohol, calorie, and pH measurements between the two treatments when comparing each yeast species were highly significant (p < 0.01), and less so when measuring RDF (p < 0.05). Dryhopping has been shown to biochemically change the composition of wort during fermentation, allowing yeast access to a greater amount of fermentable sugars and subsequent additional fermentative capacity, a phenomenon known as hop creep [80][81][82][83][84]. Most of the novel yeasts shown here show no ability to mitigate the hop creep phenomenon in an effective manner, as all yeasts, with the exception of S. mikatae UCDFST 11-510, showed increases in RDF ( Table 3) and alcohol (Fig. 5) from the addition of dry-hops during fermentation.  Fermentation kinetics were grouped more closely in the dry-hopped fermentations compared to the non-hopped treatment (Fig. 5) (Fig. 5b), but was still a slower fermenter than the control strains in both treatments. The S. Of note, is the strain UCDFST 11-510, S. mikatae, as it was an outlier from the group with the lowest RDF ( Table 3) and final amount of alcohol produced, whether dry-hopped or not (Fig. 5). UCDFST 11-510 recorded 99.0 ± 0.5% yeast viability in suspension at the end of fermentation, yet only 14.2% RDF in the non-hopped treatment. This indicates the strain is a potential candidate for low or no alcohol beer fermentations if brewing parameters are adjusted to get the final alcohol below 0.5% (v/v) and considerations are taken for microbial stability.
Analysis of the sugars remaining in this beer may aid in determining which carbohydrates this S. mikatae strain was able to assimilate during fermentation. Additionally, this species has been shown to form a pellicle on top of fermenting beer after twenty-five days at 20 ºC [43], suggesting it may ferment comparably slowly as wild-type yeasts, such as Brettanomyces or Hanseniaspora spp. Further research regarding S. mikatae in fermentation for the production of low and no alcohol beers should be performed.

b. Sensory Analysis
The flavor of the beers from these fermentations was investigated for aroma, taste, and mouthfeel in order to further qualify the brewing potential of these novel Saccharomyces yeasts.  Table 2). DraughtLab software was used to confirm that statistically significant differences were observed for all of the consensus CATA terms after accounting for both panelist and replication effects. Body, Alcohol, and Astringency were selected as the most common mouthfeel descriptors.
From the panelists at participating breweries, all beers showed increases in bitterness and astringency from the high level of dry-hopping (Fig 6), suggesting beer clarification prior to packaging may have been necessary to fully distinguish the effects of the hops without particulates in suspension effecting flavor. The base beer was also of low alcohol and IBU content, which could contribute to perceived bitterness from the increase of humulinones from dry-hopping a low IBU beer [85], or perceived astringency from the increase of polyphenol content [80]. Dry-hopping increased the fruit (Citrus, Tropical, and Stone Fruit) perception on all beers as expected from the Centennial cultivar used here [86], with the exception of Stone Fruit in UCDFST 01-161 S. paradoxus.
All experimental beers displayed Spicy aromas, likely from the expression of phenols, but genetic testing for the POF phenotype should be performed to confirm [87]. Interestingly, these Other descriptors were written in on the ballot ( kudriavzevii UCDFST 11-515 were described as having distinct phenolic and sulfur characteristics in the non-hopped treatment. The non-hopped beer fermented with S. mikatae UCDFST 11-510 was perceived as being wort-like, likely due to its low attenuation. Descriptors are given only if more than 10% of panelists (n = 5) reported a given characteristic.

D. Conclusion
Fermentation kinetics and yeast viabilities here suggest appropriate pitching rate, adequate nutrients, and proper aeration from the brewhouse were achieved on all brews and fermentations. All yeasts reached terminal gravity in under two weeks, with the exception of S.
uvarum UCDFST 11-512, which took fifteen days for the non-hopped fermentation. These kinetics makes all the yeasts studied viable candidates for production breweries, but conditioning time should be accounted for but were not studied here. All fermentations in this study were deemed terminal based on gravity as opposed to metabolite production, so further analysis and brewer-specific standards are required. All yeasts displayed high potential for re-pitching in a commercial setting with high viabilities at the end of fermentation in the non-hopped fermentations. These high numbers are promising, but viability should be assessed during fermentation and prior to re-pitch in order to ensure adequate cell count for vigorous growth in a commercial setting.
With the exception of S. mikatae UCDFST 11-510, all yeasts displayed increased RDF and alcohol with the addition of dry-hops during fermentation, as was expected due to hop creep.
Further research should be pursued in the use of S. mikatae  and other strains of this species for the production of low and no alcohol beers and its possible resistance to hop creep. Strong phenolic characteristics were perceived in the flavor of beers fermented with all yeasts, but dry-hopping, in this case with Centennial, decreased this aroma while increasing all fruit aromas, as well as bitterness and astringency. No flavors that are generally associated with poor fermentation scored high among trained sensory panels. Comparisons to standard beer yeast fermentations should have been performed in sensory analysis as well, but experimental design mistakes and time constraints did not allow. Previous research has shown these yeasts' ability to co-ferment with standard S. cerevisiae, and flavor analysis should also be performed on these potential combinations. All of these species in the Sss displayed great brewing potential given a brewery's desire to experiment with flavor and willingness to bring in a new yeast.