Article

Alternative Souring Methods: Acid-producing yeast strains

Grape skins contain an amazing diversity of microorganisms that scientists and yeast ranchers are exploring for various purposes. Photo by Wes Hagen

In recent years, sour beers have risen in popularity thanks to the niche of consumers who find the array of fruited or barrel-aged or mixed fermentation tart beverages an exciting new world to explore. In Europe, sour beers are traditionally made with an obscure brewing process that has been utilized for centuries. They have the right complexity to take off in a market obsessed with the in-depth search for layers of complexity. Sour beers begin with the same mashing technique as all beers, but then a variety of microorganisms impart the tart taste to the drink and also produce different combinations of flavors each time, ranging from fruity to funky. Brews that would have been thrown away in the recent past, are now requiring more and more tank space in the breweries, and old traditional recipes have been twisted by experienced brewers to keep up with the growing demand.

The Newest Innovation in the Production of Sour Beers

The category of sour includes different types of beers with a common sour taste and low pH due to organic acid concentration (mainly lactic and acetic). These beers are generally fermented using both bacteria and yeasts. Traditionally souring is achieved by fermentation followed by a long-term aging process brought forth by a consortium of indigenous or inoculated microbes. This approach has several limitations, primarily poor process control, lack of consistency in product quality, long processing time, and risk of cross-contamination. Kettle souring has therefore become a common alternative to traditional souring. It involves inoculation and acidification by specific lactic acid bacteria (LAB) in the brewing kettle, followed by a second boiling executed with the purpose of eliminating the bacteria. Only then, wort is transferred and fermented with yeast. While this newer method enables quick souring, the resulting beers generally lack depth and complexity due to this extra boiling, which causes the loss of aroma compounds and is responsible for the absence of the specific characteristics that originate from the metabolic activities that occur during the souring process. Moreover, this technique, when applied on a commercial-scale, can block further brewing in so far as the kettle cannot be used for production of new wort.

The use of yeast that both acidifies and ferments (referred to as “primary souring” because it is executed by the same organism during primary fermentation) represents an interesting alternative that needs an in-depth investigation. The process can be simplified and shortened without the addition of bacteria, thereby reducing the risk of cross-contamination. In addition, aroma compounds are retained in this souring method.

Many potential non-Saccharomyces yeast species have been evaluated, but a yeast utilized in the wine world called Lachancea thermotolerans, whose hallmark is its acidification properties, positioned it as the most viable alternative to lactic acid bacteria for the production of sour beers. Beer fermentation by L. thermotolerans has proven to be achievable without detrimental effects on the sensory and physical properties of beer. However, according to recent studies, there is a significant variation between various strains of L. thermotolerans in fermentation performance, lactic acid production, and in the overall aroma profile. More experiments are needed to determine the influence of this non-Saccharomyces yeast on the overall aroma profile of beer.

Instead of exploring biodiversity, a different approach to creating alternative souring organisms using genetic modification resulted in a S. cerevisiae strains capable of producing lactic acid during fermentation. Sourvisiae from Lallemand and Galactic from Berkeley Yeast are two such strains. Sourvisiae is reported to produce very acidic beer (final pHs in the range of 3.0) and to finish fermentation in approximately five days; however, it imparts only a slightly fruity flavor, lacking the sought after aromatic expression. The yeast also “commits suicide” by producing too much lactic acid, therefore it cannot be reused. Final pH and ability to repitch can be mitigated by co-inoculating Sourvisiae with a regular S. cerevisiae brewing strain. Galactic yeast finishes in the 3.5–3.8 range and is reported to be re-pitchable.

A quick background on Lachancea thermotolerans

The biotechnological potential of yeasts other than Saccharomyces, commonly referred to as “non-conventional” or “non-Saccharomyces” brewer’s yeasts, has triggered the scientific interest to untap one remarkable species: Lachancea thermotolerans. This yeast is commonly found on grapes and in wines worldwide and its oenological performance has been widely studied. Among yeast genera, L. thermotolerans displays a unique ability to produce lactic acid concomitant to alcoholic fermentation, and for winemakers it has proven capable of imparting positive effects on wine chemical and sensory profiles when co-inoculated with S. cerevisiae.

The key enzyme implicated in lactate biosynthesis is lactate dehydrogenase (LDH; E.C. 1.1.1.27). In S. cerevisiae, the major metabolic flux converts pyruvate from glycolysis to ethanol: The fermentation to lactate may also provide an alternative pathway that, similarly to alcoholic fermentation, fulfills the cellular redox balance (Figure 1). The molecular mechanisms underlying this lactate production in L. thermotolerans at the expense of ethanol or other metabolites are still poorly understood. Depending on the inoculated strain and the fermentation conditions, different strains of L. thermotolerans may in fact produce different concentrations of lactic acid during wine fermentation (1.0 and 16.8 g/L in L. thermotolerans monocultures; in mixed fermentations with S. cerevisiae 0.18 to 6.38 g/L).
This peculiar feature of L. thermotolerans has led to the commercialization and the adoption of various active dry yeast (ADY) strains in the wine industry: Laktia (Lallemand), Concerto (Chr. Hansen), Octavia (Chr. Hansen), Levulia Alcomeno (AEB), Zymaflore Omega (Laffort) and Excellence X-FRESH (Lamothe-Abiet). However, the process of biological acidification can be used in wine must (unfermented grape juice) and also in brewer’s wort for obtaining a rapid drop in pH. As a result, last year a novel Lachancea spp. strain was found in nature by the University of the Sciences in Philadelphia and been brought into production with the sole purpose of sour beer production (WildBrew Philly Sour by Lallemand). The yeast is reported to produce moderate amounts of lactic acid, typically resulting in a final pH range of 3.2–3.5 and to complete fermentation in 10 days, if proper inoculation and certain fermentation parameters are met. The bouquet includes red apple, stone fruit, and peach.

Figure 1: Lactic Acid Production. Lactic acid production by L. thermotolerans. Lactic acid dehydrogenase (LDH) converts pyruvate to lactic acid (with NAD+ regeneration).

The search for a novel L. thermotolerans

Four years ago, from a 5-gallon (19-L) batch of homebrew with friends and former colleagues at the University of Adelaide, I began my deep dive into the brewing characteristics of L. thermotolerans. To develop a reliable production method for commercial use that would represent an alternative to bacteria, an initial screening of more than twenty multiple strains (commercially available and from yeast banks) was performed. Upon characterization, only a few L. thermotolerans showed potential for beer fermentation, exhibiting good fermentation performance, sugar and nitrogen compound utilization, resistance towards higher ethanol levels and hop antimicrobial iso-α-acids, altogether with considerable lactic acid production. A diverse output from L. thermotolerans strains in malt extract was observed, ranging consistently higher final gravity compared to S. cerevisiae (i.e., different ability to consume maltose and to not use maltotriose) and a pH ranging from 3.4 to 4.0.

One strain of particular interest was “BBMCZ 7FA20,” which I’ll reference as FA, an indigenous yeast isolated from grape skins in the Burgundy region of France whose fermentation process resulted in lower final pH compared to S. cerevisiae (SC), but not excessively tart, as pH 3.2 can sometimes be perceived. However, due to the longer transformation of sugars into alcohol, as well as the relatively high quantity of unfermented sugars left in the final product, the yeast resulted as being characterized better in mixed fermentation with SC. FA was sequentially inoculated with SC at different times (after 2 and 7 days) and compared to SC monoculture in pilot-scale ale beer fermentations (OG 1.038; initial pH 5.3). The FA sour beers reached pH 3.5 while SC remained at 4.1 (Figure 2). Lactic acid production averaged at 2.5 g/L (Table 1). An expert panel comprised of brewers and certified beer tasters confirmed the absence of faults. The mixed culture fermentation with FA produced a distinct aroma profile compared to the aroma produced with monoculture control. The panel described the SC beers as “not sour,” “fruity,” “phenolic,” and “spicy,” while FA beers were picked up as “sour,” with “citrusy” and “fruity” aroma.

Figure 2: Fermentation and pH Profile
Table 1: Analytical Parameters of the Final Beers

General Fermentation Characteristics

Sugar consumption and lactic acid production

FA has interesting fermentation dynamics. Fermentation starts rapidly before the pH drops to 3.5 +/- 0.2 in 48–72 hours (Figure 4). An inoculation rate of 1 x 107 cells/mL is recommended to correctly start fermentation (= 100 g/hL or 4.3 oz./BBL, similar to standard ale yeast pitching rates) and maximize lactic acid production. Furthermore, given the fact that dry yeast comes pre-oxygenated there is no need for additional oxygenation at the beginning or throughout the fermentation. An additional dose of small glucose or low mash temperature may also be adopted to improve lactic acid production. Because of the limited sugar consumption, FA inoculation must be followed by another yeast (sequential inoculation). Depending on the specific fermentation condition, two to three days is the best time to inoculate the regular S. cerevisiae and finish fermentation. The yeast sequentially inoculated shows a fermentation rate slower than normal due to the lack of glucose in the fermenting wort. FA is compatible with any ale, lager, saison, and Brettanomyces yeasts, depending on the desired type of beer.

Flavor Profile

Fresh and fruity aromatic profiles can be obtained depending on the temperature of fermentation: Grapefruit at a relatively cool temperature (64 °F/18 °C) and tropical expressions at a higher temperature (75–86 °F/24–30 °C). No Band-Aid-like or “rotten eggs” flavors, which are commonly associated with the presence of bacteria or with problematic fermentation, can be perceived.

Repitching and microbial control

There is no risk of cross-contamination due to the limited sugar consumption and the fact that this strain is neutralized by cell-to-cell contact by any S. cerevisiae. This process not only allows for the maintenance of CIP (clean-in-place)cleaning standard procedures, but also for a higher control of beer acidification. If a higher pH is desired, early inoculation of S. cerevisiae results in an early stop of the acidification (allow 12 hours for S. cerevisiae to take control of the fermentation). For the same reason, however, no repitching of the cone is possible, insofar as the FA cells are poorly viable at the end of fermentation.

Figure 3: Plato Measurements
Figure 4: pH Measurements

Conclusion

The number of sour beers has increased substantially in recent decades, sparked by the growing interest of consumers for new flavors. The traditional approaches to sour beer production and fermentation result in complex products with numerous positive sensory properties, but such approaches present several logistical (and cash flow) issues for brewers.

The desire to overcome these issues has contributed to the development and application of innovative materials and non-Saccharomyces yeasts in the process of brewing. The screening and selection of new species has unveiled the potential of some L. thermotolerans strains based on their potential for biological acidification and flavor generation. Along with the WildBrew Philly Sour, “BBMCZ 7FA20” appears adequate to produce appealing sours in sequential inoculation with regular yeast, and it has been commercialized for the production of sour beers (Fermobrew Acid, AEB). It’s an exciting new world for brewers to explore.

References

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Issue: October 2021