Food Research International 86 (2016) 112–120

Contents lists available at ScienceDirect

Food Research International

journal homepage: www.elsevier.com/locate/foodres

Review

Could non-Saccharomyces yeasts contribute on innovative

brewing fermentations?
Rafael Felipe Basso, André Ricardo Alcarde, Cauré Barbosa Portugal ⁎
Dept. Agroindústria, Alimentos e Nutrição, Escola Superior de Agricultura “Luiz de Queiroz”, University of São Paulo, Avenida Pádua Dias 11, 13418-900 Piracicaba, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: With the advances in the production of beer worldwide, more challenges arise each year in the search for new
Received 24 April 2016 approaches to the development of distinctive beverages. Attempts to obtain products with more complex senso-
Received in revised form 3 June 2016 ry characteristics have led experts and brewers to prospect for non-conventional yeasts, i.e., non-Saccharomyces
Accepted 4 June 2016
yeasts that may provide a new range of perspectives in terms of techniques and approaches. Besides the wide-
Available online 6 June 2016
spread use of Dekkera/Brettanomyces for the production of sour beers, other species are emerging for presenting
Keywords:
unusual metabolic features that include the production of fruity esters, and a distinctive enzymatic apparatus.
Non-conventional yeasts Wickerhamomyces anomalus and Torulaspora delbrueckii stand out as the most promising yeasts in brewing pro-
Brewing cesses. Such new tendencies in the use of non-Saccharomyces yeasts comprise the production of low-alcohol
Specialty beers beers, functional beers, and bioflavoring approaches. This is a little explored field in brewing practice, still requir-
Low-alcohol beers ing extensive research with practical application. In this sense, this review aims to present the main points for the
Biotechnology application of non-conventional yeasts in beer production, and thus contribute to future advances in the topic.
Bioflavoring © 2016 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
2. Non-Saccharomyces yeasts in brewing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
2.1. Dekkera/Brettanomyces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
2.2. Other yeasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
2.2.1. Wickerhamomyces anomalus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
2.2.2. Torulaspora delbrueckii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
3. Technology and product innovation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
3.1. Recovering old production methods for innovation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
3.2. Bioflavoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
3.3. Low calorie beers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
3.4. Low alcohol beers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
3.5. Functional beers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
4. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

1. Introduction
The brewing of beer-like beverages already occurred between 5500–
3100 BCE in the regions of Mesopotamia and Ancient Egypt (Hornsey,
⁎ Corresponding author.
E-mail address: caure.portugal@gmail.com (C.B. Portugal).
2007). Barley grew in wild forms in these regions, and such civilizations
found that moistening, germinating, and drying the grains would lead to
a sweeter, less perishable product (Papazian, 2003). This is what we
now perceive as barley malt. Archaeological evidences suggest that in
such times beer was made from barley, wheat, or a mixture of both.
The process was carried out by spontaneous fermentations, either by
preparing a cereal slurry, or by using a mixture of raw grains, cooked

http://dx.doi.org/10.1016/j.foodres.2016.06.002
0963-9969/© 2016 Elsevier Ltd. All rights reserved.
 R.F. Basso et al. / Food Research International 86 (2016) 112–120 113

malt and water, with further exposure to ambient air to induce “con- appear. However, many of them are already used as pure inoculum, or
tamination” (Sicard & Legras, 2011). in yeast blends under controlled conditions of processing for specialty
Since the species of the genus Saccharomyces – especially Saccharo- beer production (Hayashi, Arai, Tada, Taguchi, & Ogawa, 2007).
myces cerevisiae – frequently appeared as abundant and dominant In most beers, the microorganisms that carry out fermentation be-
agents in spontaneous fermentations, they were intuitively selected long to different Saccharomyces species and strains (Bokulich &
over generations, in different cultures and from distinct raw materials. Bamforth, 2013). The occurrence of other species is commonly reported
Readily, men figured out that using a small piece or volume of a modi- in some peculiar beer styles produced by spontaneous fermentations, as
fied product into a fresh one would work as a starter for inducing fer- the Belgian Lambics and Gueuzes, and their American counterparts,
mentation again. It was only in the mid 17th century scientists began Coolship Ales. However, due to the diversity of substrate assimilation
to understand what was behind fermentation, enabling the isolation patterns that non-conventional yeasts may display (van Dijken, 2002),
techniques, selection and use of pure inocula in those processes the use of non-Saccharomyces in controlled fermentations has been
(Steensels & Verstrepen, 2014). Successive investigations, that included gaining popularity among brewers in order to obtain distinctive prod-
yeast's description by Antonie van Leeuwenhoek in 1680, its association ucts, a key point to gain market share in this growing segment (Ciani
with beer wort fermentation by Cagniard de Latour in 1836, the estab- & Comitini, 2011; Cordero-Bueso, Esteve-Zarzoso, Cabellos, Gil-Díaz, &
lishment of isolation techniques, and the use of pure starters as top or Arroyo, 2013; González, Quirós, & Morales, 2013; Johnson, 2013). To
bottom fermenting yeasts by Emil Hansen in 1883, enabled the im- take one example, the market of craft beers in the United States grew
provement of controlled and industrial processes (Lodolo, Kock, 12.8% in 2015, increasing 16.3% in exports, and with incomes of 22.3 bil-
Axcell, & Brooks, 2008). lion dollars, opening 620 craft breweries – only 68 closings in the same
There are many different styles of beer but, primarily, the main period – and establishing a goal of achieving 20% of beer market share
brewing classification criterion particularly relies on the type of fermen- by 2020 (Brewers-Association, 2016; Kell, 2016). A significant strength-
tation. Although this concept may vary, brewing yeasts are mainly clas- ening of the sector has also been detected in several European countries,
sified as top-fermenting or ale yeasts (Saccharomyces cerevisiae), and where growth sometimes outstripped 300–1000% from 2009 to 2014
bottom-fermenting or lager yeasts (Saccharomyces pastorianus). The (Table 1).
two main styles of beer are still based on such traits, of which top- The production of quality beer relies on the activity of fermenting
fermenting yeasts display more genetic divergence, and the process yeasts that are qualified not only for good fermentation yield-efficiency,
normally occurs between 18 °C – 24 °C, while bottom-fermenting yeasts but also for printing aroma and flavor to the beverage. Their importance
show a conserved genetic material, and often ferment at lower temper- in beer production goes beyond the formation of ethanol and carbon di-
atures (8 ° C – 14 ° C) (Lodolo et al., 2008). Fermentations for Lambic oxide, since most of the compounds that provide taste and aroma arise
beers, on the contrary, is a spontaneous, uncontrolled process, driven during fermentation, and these are intermediate compounds and by-
by brewery-resident microorganisms that are introduced by exposing products of metabolism (Pires, Teixeira, Brányik, & Vicente, 2014).
the wort in shallow tanks during the overnight cooling, before transfer- In this sense, this review aims to recover and discuss recent informa-
ring it to wooden barrels for fermentation and aging. tion on the status of non-conventional yeasts, and their potential appli-
Since the establishment of sedentary life by mankind, and formation cation in beer production. Even so, this work intends to present the
of the first human social structures, multiple events – including natural main technological approaches regarding the use of non-Saccharomyces
and human selection – led to a pool of domesticated or selected yeasts as a way to improve sensory traits, as well as product diversification and
(Sicard & Legras, 2011). Selected Saccharomyces strains are used with innovation in brewing processes.
various purposes because of their plasticity on assimilating different
substrates, usually not incorporated by S. cerevisiae. The use of Saccharo- 2. Non-Saccharomyces yeasts in brewing
myces strains in controlled fermentations over decades is essentially
based on three main features: (a) efficient production of high ethanol Saccharomyces cerevisiae is the widespread brewing yeast, and se-
amounts; (b) the use of fermentation as the preferential metabolic lected strains have proportionated improvement and control of pro-
pathway, combined to the positive Crabtree effect (repression of respi- cesses as a way to obtain chemical and sensory quality, as well as to
ration by glucose); and (c) higher tolerance to ethanol and other envi- maintain reproducibility. The expansion in the sector, and the growing
ronmental stresses (Steensels & Verstrepen, 2014). number of specialized consumers, have driven brewers to innovate
Nonetheless, with increasing demands for innovative products, methods changing hop varieties, using special malts, preparing distinc-
other non-Saccharomyces species have been isolated and characterized tive blends, and more. Nevertheless, it is in the soul's beverage, in the
for the development of specialty beers, with distinctive aromatic and fermentation and pitching non-conventional yeasts, where producers
flavor components (Vanderhaegen et al., 2003). They generally present are finding not only a multiplicity of aroma and flavor production, but
low fermentation yields, and are more sensitive to ethanol stress (Di also new approaches and impressions in the overall organoleptic profile
Maro, Ercolini, & Coppola, 2007), but may display a great range of pos- of beers (Michel et al., 2016). In this sense, some old acquaintances and
sibilities for beer fermentations in order to provide distinctive aroma
and flavor (Renouf & Lonvaud-Funel, 2007). It has been shown, for ex-
Table 1
ample, that Dekkera bruxellensis and Hanseniaspora uvarum (anamorph,
Number and growth of microbreweries in Europe (2009–2014).
Kloeckera apiculata) have the ability to produce many esters with fruity
character, besides promoting the enzymatic breakdown of some pro- Microbreweries
teins (De Keersmaecker, 1996; Kumara & Verachtert, 1991). Lately, 2009 2014 Relative growth (%)
other wild yeast species have been mentioned as interesting alterna-
Spain 27 314 1062
tives in brewing by their genome particularities, as in the case of D. Norway 13 65 400
anomala, Hanseniaspora uvarum, S. bayanus (= S. uvarum), S. Sweden 30 149 396
pastorianus, Naumovozyma dairenensis (=Naumovia dairenensis), and Czech Republic 51 238 366
Slovakia 9 39 333
Debaryomyces spp. These microorganisms can display unusual metabol-
Slovenia 20 49 145
ic abilities, thus changing the sensory character of the final product by Italy 242 585 141
adding complexity in flavor, and modifying mouthfeel sensation France 263 566 115
(Spitaels et al., 2014). Some yeasts are eventually characterized as spoil- United Kingdom 694 1414 103
age agents, as in the case of Dekkera/Brettanomyces and Debaryomyces – Switzerland 232 440 89

especially in poorly controlled processes in which they unintentionally Source: Beers Statistics 2015 (The Brewers of Europe).
114 R.F. Basso et al. / Food Research International 86 (2016) 112–120
other promissory microorganisms have outlined an ever-increasing
range of techniques and sensory results in brewing.
In Table 2 we summarize the main yeast species discussed in this
work, presenting their current taxonomical status, major isolation
sources recorded in literature, as well as their food safety classification.
Additionally, Table 3 brings most prominent information about their
major aromatic compounds produced and descriptors, in addition to
the related enzymes and peculiar traits of each species. In the following
topics, we discuss their specific characteristics, and thereafter, some
technology and product innovation regarding the use of these non-con-
ventional yeasts.
2.1. Dekkera/Brettanomyces
In the early 1970s, almost all known species of Dekkera/
Brettanomyces had been isolated from spontaneous brewing processes.
Today, they are the most relevant non-conventional yeasts in the pro-
duction of sour beers, mainly by the discovery of its singular influence
in Lambic beers, and have attracted growing interest from brewers
and connoisseurs. Lambics are produced under a complex succession
of spontaneous fermentations, in which Dekkera/Brettanomyces yeasts
take part during the acidification-maturation stage, a process occurring
after three or four months after fermentation starts (Spitaels et al.,
2014). This final maturation phase may last up to ten months, and it is
known that these yeasts are responsible for producing a set of flavor
compounds, mainly ethyl phenol, ethyl guaiacol, isovaleric acid, acetic
acid, and ethyl acetate, which together result in a typical flavor charac-
ter (Vanderhaegen et al., 2003; Verachtert & Derdelinckx, 2014). How-
ever, an increasing number of researches addressing to the use of
these yeasts in controlled fermentations has been observed in the last
years, both in pure cultures as in co-inoculation with S. cerevisiae
(Steensels & Verstrepen, 2014).
Beyond their typical phenolic notes, those commercial Dekkera/
Brettanomyces strains have shown the ability to exert a marked influ-
ence on the sensory profile, contributing to an overall fruity or floral
character by producing high levels of ethyl esters (ethyl acetate, ethyl
caprate, ethyl caprylate, ethyl lactate) (Crauwels et al., 2015). It is note-
worthy that some peculiar flavors – namely “horse blanket”, “stable”
character – may also arise, and in small concentration, account for a pos-
itive and complex bouquet. On the other hand, misguided fermenta-
tions with numerous Dekkera/Brettanomyces communities may lead to
some unpleasant off-flavors that include “acrid smoke”, “band-aid”
and “fecal” descriptors. The unique taste and aroma traits they are
able to imprint is a fact particularly related to the activity of certain
Table 2
Taxonomical status, isolation sources and use of non-conventional yeasts for brewing.
Species Anamorpha Synonymsa
Cyberlindnera saturnus Williopsis saturnus; Saccharomyces saturnus
Dekkera anomala Brettanomyces Dekkera/Brettanomyces claussenii
anomalus
Dekkera bruxellensis Brettanomyces Brettanomyces lambicus; Brettanomyces
bruxellensis custersii
Hanseniaspora uvarum Kloeckera apiculata Kloeckeraspora uvarum; Hanseniaspora
apiculata
Pichia kluyveri Hansenula kluyveri
Torulaspora delbrueckii Candida colliculosa Saccharomyces rosei; Saccharomyces delbrueckii
Wickerhamomyces Pichia subpelliculosa; Hansenula subpelliculosa
subpelliculosus
Wickerhamomyces Candida Pichia anomala; Saccharomyces anomalus;
anomalus beverwijkiae Hansenula anomala
a
Taxonomical records according to National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/guide/taxonomy), and Index Fungorum (http://
www.indexfungorum.org).
b
Data from Global Catalogue of Microorganisms (http://gcm.wfcc.info).
c
According to Bourdichon et al. (2012).
amylases, although it a strain-dependent trait (Daenen, Sterckx,
Delvaux, Verachtert, & Derdelinckx, 2008).
Since its first description in 1904 by Niels Hjelte Claussen, director of
the Carlsberg Brewery at that time, there were several taxonomic
changes for the genus. Currently, Dekkera is the official nomenclature,
adopted for the sexual form or teleomorph, and Brettanomyces desig-
nates the anamorphic or asexual form of this yeast (Kurtzman, 2011).
D. bruxellensis and D. anomala are the species that typically appear in
brewing processes (Steensels et al., 2015; White & Zainasheff, 2010).
Besides presenting certain characteristics that enable their good perfor-
mance in alcoholic fermentations, including tolerance to osmotic pres-
sure, ethanol, limiting oxygen and low pH, these species show a
diverse metabolism that leads to interesting and unusual results in
beer production (Steensels et al., 2015).
When compared to S. cerevisiae, these yeasts have greater genetic di-
versity, and may present higher gene doses due to increased ploidy var-
iability, especially in low-oxygen and high sugar concentration
environments (Piškur et al., 2012; Woolfit, Rozpędowska, Piškur, &
Wolfe, 2007). D. bruxellensis and D. anomala usually display the Crabtree
effect, promoting fermentation instead of respiration, even with avail-
able oxygen in media with a certain concentration of sugars (Thomson
et al., 2005). However, the metabolic trait that markedly differentiates
them from those traditional yeasts is the expression of the Custer effect.
In this case, these organisms have greater ability to conduct the fermen-
tation in the presence of oxygen by reducing or ceasing the glucose me-
tabolism (lag phase) under anaerobic conditions (Barnett & Entian,
2005). It is worth mentioning that small oxygen inputs during fermen-
tation is encouraging to best results in biomass and ethanol production,
fermentation efficiency, total consumption of glucose, and low produc-
tion of acetic acid (Aguilar-Uscanga, Délia, & Strehaiano, 2003).
Dekkera/Brettanomyces yeasts are able to metabolize a wide range of
carbon sources, including cellobiose and dextrins. These sugars are not
readily assimilated by Saccharomyces species, except in the case of S.
cerevisiae var. diastaticus, which is able to metabolize dextrin and starch
(Steensels et al., 2015). Cellobiose is a disaccharide found in wooden
barrels used to mature beers (Daenen, Sterckx, et al., 2008;
Vanderhaegen et al., 2003), and dextrins are more complex sugars
that include maltotetraose and maltopentaose. Such molecules are the
main components of the residual sugar content in beers fermented by
S. cerevisiae, and can be degraded by the β-glucosidase. This enzyme,
commonly produced by Dekkera/Brettanomyces, enables degradation
of those complex molecules into simple sugars that can be readily as-
similated, resulting in a “super-attenuated” beer. The aforementioned
methodology includes some particular traits, and practical results in
Food
Isolation sourcesb Application safetyc
Insect, soil Bioflavoring; low Safe
alcohol beer
Beer, cider, soft drink Bioflavoring; low Safe
calorie beer
Beer, grape, wine, ginger ale Bioflavoring; low Safe
calorie beer
Grape must, fruits, soil, sour dough, Bioflavoring Safe
insect, flowers
Olives, fruits, flowers, soil Bioflavoring Safe
Beer, fruits, soil, milk, moss, mushroom Bioflavoring; low Safe
alcohol beer
Fruits; vegetables; molasses; jam; milk Low alcohol beer Safe
Beer, sour dough, fruits, sake, wine, Bioflavoring; low Safe
oak calorie beer
 R.F. Basso et al. / Food Research International 86 (2016) 112–120 115

Table 3
Major aromatic compounds, enzymes and peculiar traits of non-conventional yeasts in brewing.

Flavor Aromatic Prominent

Species compound descriptor enzyme Disadvantage Peculiar traits References

C. saturnus Isoamyl Banana, AAT Biofilm formation Ethanol sensitive Erten & Campbell (2001), Erten & Tanguler (2010), Lee et al.
acetate pear (2010)
Ethyl acetate Fruity,
Solvent
D. anomala Acetic acid Vinegary BGL, CDC, High acetic acid Osmotolerant; pH Lentz & Harris (2015)
Ehtyl Clove; VPR production; Overall tolerant; Custer effect
phenols Leather off-flavor
Ethyl esters Fruity
D. bruxellensis Acetic acid Vinegary BGL, CDC, High acetic acid Osmotolerant; pH Lentz & Harris (2015)
Ehtyl Clove; VPR production; Overall tolerant; Custer effect
phenols leather off-flavor
Ethyl esters Fruity
H. uvarum Isoamyl Banana; BGL Cycloheximide-resistant; Arévalo Villena, Úbeda Iranzo, Cordero Otero, & Briones Pérez,
acetate pear Ethanol tolerant (2005), Spitaels et al. (2014), Wang, Mas, & Esteve-Zarzoso
Ethyl acetate Fruity; (2015)
solvent
P. kluyveri Isoamyl Banana; AAT Osmotolerant; Pasteur Fredlund (2004), Ma, He, Cao, Bai, & Li (2016), Sarens &
acetate pear Effect; pH tolerant Swiegers (2014), Steensels & Verstrepen (2014), Walker (2011)
Isobutyl Banana;
acetate fruity
T. delbrueckii Isoamyl Banana; AAT, EST, High acetaldehyde Osmotolerant; Canonico et al. (2016), Charoenchai, Fleet, Henschke, & Todd
acetate pear INV, BGL production cryotolerant; ethanol (1997), Peinado, Moreno, Bueno, Moreno, & Mauricio (2004),
Ethyl Banana; tolerant Plata et al. (2003)
butyrate apple;
acetaldehyde strawberry
green apple
W. Isoamyl Banana, AAT, EST Biofilm formation; (Erten & Campbell (2001), Plata et al. (2003)
subpelliculosus acetate pear excessive ester
Ethyl acetate fruity; production
solvent
W. anomalus Ethyl acetate Fruity; EST, BGL Biofilm formation Ethanol sensitive; Charoenchai et al. (1997), Erten & Campbell (2001), Passoth et
solvent Osmotolerant; pH al. (2006)
tolerant

Alcohol acetyltransferase (AAT), Esterase (EST), Invertase (INV), β-glucosidase (BGL), Cinnamate decarboxylase (CDC), Vinylphenol reductase (VPR).

beer styles in which low calorie and residual sugar content constitute
innovative hot spots (Kumara, De Cort, & Verachtert, 1993; Steensels
et al., 2015).
Regarding the influence of temperature on fermentations driven by
Dekkera/Brettanomyces, most studies focus on biomass production rate,
primary products (ethanol, acetic acid and carbon dioxide), and glucose
consumption patterns. Fermentations processed between 20 °C – 32 °C
result in better ethanol production, as well as lower acetic acid contents
released in the medium (Brandam, Castro-Martínez, Délia, Ramón-
Portugal, & Strehaiano, 2007). High concentration of these compounds
can directly affect cell viability, aptitude in bioconversion of substrates,
and low fermentative capacity. Besides disturbing yeast growth and lead-
ing to acidic shock conditions, intense release of acetic acid also increases
the risk of impairing the volatile acidity and contribute to sensory imbal-
ance in the final product (Rogers, Veatch, Covey, Staton, & Bochman,
2016).
Although the tolerance to ethanol is a strain-dependent factor, D.
bruxellensis is noticeably resistant to concentrations of up to 13.5–
15.0% (v/v) (Barata et al., 2008), although in vitro tests show that
some strains can tolerate up to 20% ethanol (Portugal et al., 2014). D.
anomala, otherwise, is more sensitive to its toxicity, tolerating at most
8% (v/v) ethanol in the medium (Barata et al., 2008). In any case, the re-
sponse of these microorganisms to ethanol stress entails on the exten-
sion of the lag-phase, with direct consequences on growth rate and
cell density, in addition to increased concentrations of ethyl phenols
and ethyl esters in the medium (Conterno, Aprea, Franceschi, Viola, &
Vrhovsek, 2013).
The production of acetic acid by Dekkera/Brettanomyces is part of the
strategy based on “make-accumulate-consume”. Such system involves
an interesting evolutionary feature that gives it a competitive edge, char-
acterized by the production and accumulation of ethanol and acetic acid
in the fermentative environment, with subsequent degradation of these
compounds when fermentable sugars are depleted (Rozpędowska et
al., 2011). Although considered a fault in many beer styles, low concen-
trations of acetic acid (b 1.2 g/l) are acceptable in those ones that use
Dekkera/Brettanomyces in their primary fermentation (Steensels et al.,
2015). Higher concentrations of this compound, however, appear to in-
hibit substrate consumption. The metabolic regulation of this by-product
is highly dependent on oxygen availability, with a direct relation between
oxygen concentration in the substrate and the acetic acid production
(Aguilar-Uscanga et al., 2003). It is known, moreover, that the tempera-
ture influences only slightly in the production of this compound
(Brandam et al., 2007).
There is a set of very peculiar sensory characteristics that Dekkera/
Brettanomyces imparts to the beverage, mainly by the production of
phenolic off-flavors (POF) that are frequently described as “rubber”,
“burnt plastic”, “medicinal”, “leather”, and “barnyard”. Much of this in-
fluence relies on the ability to metabolize hydroxycinnamic acids, of
which ferulic, p-coumaric and caffeic acids are the most common
(Chatonnet, Dubourdie, Boidron, & Pons, 1992; Lentz & Harris, 2015).
These compounds are especially present in the bark, pericarp, aleurone,
and endosperm of cereal grains, associated to the arabinoxylans struc-
tural carbohydrates, and incorporated to wort during the mashing pro-
cess (Vanbeneden, Van Roey, Willems, Delvaux, & Delvaux, 2008; Yu,
Vasanthan, & Temelli, 2001). Dekkera/Brettanomyces has two enzymes
that work to reduce such acids into ethyl phenols in two consecutive
main reactions: a) the first one takes place by the action of the enzyme
cinnamate decarboxylase, which metabolizes the acids in their respec-
tive vinyl phenols; b) the second enzyme is the vinylphenol reductase,
which reduces those vinyl phenols into ethyl phenols. (Harris, Ford,
Jiranek, & Grbin, 2009; Vanbeneden, Gils, Delvaux, & Delvaux, 2008).
The presence of these last compounds above the perception threshold
is considered undesirable in most beer styles; however, they are essen-
tial in many others, including the Belgian Lambic, Gueuze and Red
116 R.F. Basso et al. / Food Research International 86 (2016) 112–120
Flanders, the German Weizen and Rauchbier, the American Coolship
Ale, as well as the Sour and Farmhouse Ales (Lentz & Harris, 2015).
Primary fermentations using Dekkera/Brettanomyces generally lead
to beers with low content of isoamyl acetate and ethyl phenylethyl,
but instead, high concentrations of ethyl acetate and ethyl lactate
(Spaepen, Van Oevelen, & Verachtert, 1978; Spaepen & Verachtert,
1982). Moderate levels of these latter compounds are desirable, and
may contribute to the flavor complexity of beers, but higher concentra-
tions may easily impart undesirable solvent-like, buttery and medicinal
notes to the beverage (Cortés-Diéguez, Rodriguez-Solana, Domínguez,
& Díaz, 2015). Ester imbalance occurs with the degradation of acetate
esters by the esterase enzyme, a bioprocess much more efficient than
the hydrolysis of non-acetate esters (Steensels et al., 2015).
Another striking feature on Dekkera/Brettanomyces is the production
of β-glucosidase, an enzyme responsible for the hydrolysis of glycosides.
Glycosides are non-volatile compounds composed by one aromatic
molecule (aglycone) and one β-D-glucose (glycone), with possible addi-
tional sugar units attached (Winterhalter & Skouroumounis, 1997).
Such compounds are commonly found in plants, flowers and fruits,
and some of them are used in brewing, and are present in hops and in
the wood of barrels employed for maturation (Daenen, Saison, et al.,
2008). The enzymatic hydrolysis of glycosides releases the sugar mole-
cules, further metabolized, and the aglycone, which can express aromat-
ic activity, representing a poorly explored methodology to improve the
aromatic potential of beers (Winterhalter & Skouroumounis, 1997). The
β-glucosidase activity is a strain-dependent feature, but D. bruxellensis
stands out for presenting such hydrolytic action on a number of sub-
strates, emerging as a good option for fermentations in pure culture or
in co-culture with S. cerevisiae (Daenen, Saison, et al., 2008; Daenen,
Sterckx, et al., 2008).
2.2. Other yeasts
Although Dekkera/Brettanomyces is currently the most prominent
yeast in distinctive approaches, other genera and species are reported
to occur in brewing processes. Wickerhamomyces anomalus and
Torulaspora delbrueckii currently stand out as the most relevant non-con-
ventional yeasts for brewing due to their flavoring features, tolerance to
extreme environmental conditions, low production of undesirable by-
products, and frequent association with food and beverages (Canonico,
Agarbati, Comitini, & Ciani, 2016; Michel et al., 2016; Schneider et al.,
2012). Nonetheless, other yeasts of the genera Wickerhamomyces, Candi-
da, Torulaspora, Hanseniaspora, and Debaryomyces, usually occurring and
isolated from spontaneous fermentations, have also been investigated.
The most common styles in that cases include the Belgian Lambics and
the American Coolship Ale, in which several yeast species contribute to
the typical, particular characteristics of the beer (Bokulich & Bamforth,
2013; Spitaels et al., 2014).
2.2.1. Wickerhamomyces anomalus
In contrast to S. cerevisiae and Dekkera/Brettanomyces,
Wickerhamomyces anomalus (= Pichia anomala) is able to regulate the
central metabolism of carbon according to oxygen availability instead
of doing so by the glucose concentration (Crabtree-negative), increasing
the glycolytic pathway under oxygen restriction (Pasteur effect). It en-
ables the fermentative pathway in limited oxygen conditions, in
which the enzymes pyruvate decarboxylase and alcohol dehydrogenase
are activated, but rather small concentrations of ethanol are produced in
aerobic conditions (Fredlund, 2004; Passoth, Fredlund, Druvefors, &
Schnürer, 2006). W. anomalus shows a wide spectrum of sugar assimila-
tion, being able to properly metabolize hexoses (glucose, galactose,
fructose), pentoses (arabinose, xylose), disaccharides (sucrose, lactose),
polysaccharides, as well as some alcohols, organic acids, fatty acids, and
aromatic hydrocarbons (Walker, 2011). This species also displays great
physiological plasticity, being able to grow in absence of oxygen, in wide
temperature range (between 3 °C and 37 °C), and under extreme pH
values between 2.0–12.4 (Fredlund, 2004). Strains of this genus are
somewhat sensitive to ethanol and acetate (Passoth et al., 2006), but
show tolerance to high osmotic pressure, and are efficient in their
mechanisms of adaptation to stress factors (Walker, 2011).
Regarding its contribution to sensory active compounds, this species
is a good producer of ethyl propanoate, phenyl ethanol, 2-phenylethyl
acetate, and especially ethyl acetate (Passoth et al., 2006). In contrast
to S. cerevisiae, that synthesizes ethyl acetate mainly from acetyl-CoA
and ethanol by the action of alcohol acetyltransferase (Yoshioka &
Hashimoto, 1981), W. anomalus makes it mainly by reverse reaction of
esterase from acetate and ethanol (Rojas et al., 2002; Yoshioka &
Hashimoto, 1981). Such compound is metabolically important because
of its antifungal activity (Fredlund, Druvefors, Olstorpe, Passoth, &
Schnürer, 2004), besides the property of preventing toxic accumulation
of acetic acid into the cells in limited oxygen conditions (Fredlund,
2004). From a sensory standpoint, ethyl acetate directly influences on
the taste and flavor profile of the beer, lending an interesting fruity or
an unpleasant solvent-like character, depending on the concentration
values (White & Zainasheff, 2010).
Despite some evidences suggest an apparent inability to metabolize
maltose (Walker, 2011), other studies show that some wild W.
anomalus strains are indeed able to use this sugar and to grow even bet-
ter than other commercial brewing yeasts (Y.-J. Lee et al., 2011). W.
anomalus stands as a good candidate in sequential or co-inoculated fer-
mentations with other yeasts, always considering its inhibitory action
against some microorganisms (Fredlund et al., 2004; Passoth et al.,
2006; Walker, 2011).
2.2.2. Torulaspora delbrueckii
Torulaspora delbrueckii (anamorph Candida colliculosa) is a species
whose use supposedly dates back over 4000 years, and it is well studied
today in wine controlled fermentation processes because of its good
production of fruity flavors (Albertin et al., 2014). Some authors assume
that T. delbrueckii is also one of the main responsible by the fermenta-
tion of Bavarian wheat beers (Michel et al., 2016; Tataridis, Kanelis,
Logotetis, & Nerancis, 2013). These microorganisms can undergo high
osmotic pressure conditions (highly osmotolerant) (Alves-Araújo et
al., 2007; Bely, Stoeckle, Masneuf-Pomarède, & Dubourdieu, 2008),
grow well at low temperatures (cryotolerants), and curiously require
oxygen to ferment – although not displaying the Custer effect
(Alves-Araújo et al., 2007; Hanl, Sommer, & Arneborg, 2004; Salvadó
et al., 2011).
T. delbrueckii shows invertase activity, necessary for sucrose hydro-
lysis, and subsequent consumption of glucose and fructose. The ability
to consume maltose – main sugar in wort – is conditioned by the activity
of the enzyme maltase, as well as by maltose transporters also present
in this species, highlighting that such biochemical apparatus can be
inhibited by high concentrations of glucose in the medium. Interesting-
ly, the ability to metabolize the sugar maltotriose was also proven in this
microorganism. The rate and extent to which the different sugars are
consumed depend mainly on the strain, with some of them being un-
able to consume maltose and maltotriose (Alves-Araújo et al., 2007;
Michel et al., 2016).
Ethanol tolerance and fermentation efficiency are also strain-depen-
dent features. Although it has been reported that T. delbrueckii is able to
tolerate up to 11% (v/v) ethanol in winemaking conditions (Tataridis et
al., 2013), recent evidences demonstrate that many strains are only able
to grow in concentrate beer wort with at most 5% ethanol in the medi-
um (Michel et al., 2016).
Tolerance to isoα-acids from hops is another important trait that
yeast should display in brewing conditions, once that compound has in-
hibitory effect against some microorganisms, including gram-positive
bacteria and yeasts. T. delbrueckii can grow – even with an increase in
the lag-phase – in the presence of up to 90 ppm isoα-acids in the medi-
um, a concentration that correlates to highly hopped beer styles. Resis-
tance to such compounds derived from hops can be evident even in the
 R.F. Basso et al. / Food Research International 86 (2016) 112–120
presence of alcohol, since T. delbrueckii is able to grow in wort with an
ethanol content of up to 5% (v/v), and can still manage levels of up to
50 mg/l isoα-acid (Michel et al., 2016). Of course, these are strain-de-
pendent traits, but the ability to deal with some ethanol content in the
medium – toxic to the cell – provides better membrane selectivity con-
ditions. As a result, some strains of T. delbrueckii are less sensitive to an-
timicrobial effects of isoα-acids, and may be proposed as a tool for
average strength and more hoppy beers.
The most interesting contribution of this yeast is perhaps the profile
of volatile compounds it may confer to the final beer. Some recent inves-
tigations reveal the ability of some strains to produce 2-phenylethanol
(sweetish, “rose petals” and floral flavors) (Etschmann et al., 2015), n-
propanol and i-butanol, amyl alcohol (“solvent brandy” aroma) (Pires
et al., 2014), and ethyl acetate (Meilgaard, 1982). T. delbrueckii seems
to produce low amounts of isoamyl acetate (Hernández-Orte et al.,
2008), as in the case of ethyl hexanoate, ethyl octanoate and ethyl
decanoate, whose concentrations were negligible, and below the per-
ception threshold (0.01 mg/l) (Michel et al., 2016). Unlike Dekkera/
Brettanomyces, the enzymatic apparatus necessary to hydroxycinnamic
acids' reduction is not present in T. delbrueckiii, so that this species does
not have the inconvenience of producing phenolic off-flavors. The same
is not true in the case of diacetyl production, since it often releases high
amounts of this compound, which cannot be reduced during bottle or
cask conditioning. Taken as a whole, the beverages brewed with T.
delbrueckiii show honey and pear-like flavors, with highly citric and
fruity character on the palate (Michel et al., 2016).
3. Technology and product innovation
The specialty beer market has gained attention worldwide in the last
30 years. In addition to the evidence of beers produced with better qual-
ity inputs, and using methods that respect the final product attributes,
the “liquid bread” is gaining other perspectives. These new insights in-
clude the low-calorie beers, low alcohol or alcohol-free beers, gluten-
free beers, functional beers, and brewing processes that would enable
the incorporation of new flavors to the beverage (Yeo & Liu, 2014). As
well as in winemaking, brewing technology and product innovation
are greatly motivated by exploring specific traits of new yeast species.
Display of peculiar and interesting by-products or secretion of extracel-
lular enzymes, besides singular fermentative ability or sugar assimila-
tion traits, integrate some of the tools available for improving brewing
methods and results (Masneuf-Pomarede, Bely, Marullo, & Albertin,
2015). Some of these approaches simply rely on recuperating traditional
practices, but new perspectives look at bioconversion processes to en-
hance flavor characteristics, to obtain products with low calorie and/or
alcohol contents, and even being able to provide beers with current
complexions of a functional food.
3.1. Recovering old production methods for innovation
The one-process fermentation-maturation-aging in wooden barrels
was a widely used methodology in the past, probably by the lack of
choice. It is no longer extensively applied due to the growing popularity
of pure yeast cultures and stainless steel vats. Today, this procedure has
been gaining interest especially in craft breweries, representing a way of
differentiation and innovation of product. This practice renders the
micro-oxygenation of beer, besides contributing with tannins, body
and flavor complexity (Tonsmeire, 2014). Another interesting feature
of barrels is that wood actually harbor some wild yeasts, lactic acid bac-
teria, and acetic acid bacteria (Bokulich, Bamforth, & Mills, 2012;
Verachtert & Derdelinckx, 2014). These microorganisms can play a pos-
itive role on characteristics of the product, as is the case of sour beers,
where some specific metabolic traits will impart very particular com-
plexity in flavor, aroma and acidity (Spitaels et al., 2014; Tonsmeire,
2014; Verachtert & Derdelinckx, 2014).
117
Previously, bottle conditioning was the only choice for carbonating
beers until the advent of impermeable and pressure-resistant fermenta-
tion/maturation tanks, allowing a secondary fermentation and matura-
tion as well (Goldammer, 2008). Today, bottle conditioning has several
sensory advantages, with the possibility of enhancing the flavor for the
production of new metabolites, including esters and alcohols, in addi-
tion to eliminating dissolved oxygen that could contribute to precipitat-
ed deterioration of beer (Vanderhaegen et al., 2003). This is the case of
Gueuze beers, wherein young Lambics (up to one year maturation) con-
fer complex sugars to the mixture, and old Lambics (two years or more)
“inoculate” new microorganisms – especially Dekkera/Brettanomyces –
that can consume these sugars during maturation in the bottle, afford
natural carbonation, and modify the beer sensory profile (Verachtert &
Derdelinckx, 2014). In any case, it is possible to apply this technique
to most beers by adding sugars to the bottles and, optionally, pure cul-
tures of different yeast strains to increase the complexity of final prod-
uct (Vanderhaegen et al., 2003).
3.2. Bioflavoring
The appeal of consumers for beers with enhanced and differentiated
sensory profiles grows along with the search for products without
chemical additives. In this context, one of the concepts of bioflavoring
comes up for the synthesis and transformation of flavor compounds
by biological methods, including the proposal of non-conventional
yeasts (Daenen, Saison, et al., 2008; Daenen, Sterckx, et al., 2008;
Vanderhaegen et al., 2003). Addition of new compounds and flavor by
biological methods meets modern consumer expectations, either by:
(a) using pure culture of a specific yeast species; (b) sequential or co-in-
oculation with different microorganisms; (c) use of genetically modified
organisms (GMOs); or also by (d) adding isolated enzymes produced by
other microorganisms (Vanderhaegen et al., 2003). In the context of this
work, only the first two cases were considered and reviewed.
The sequential inoculation of Pichia kluyveri in beer wort is an exam-
ple, where the combined effect of different hop varieties showed to im-
prove often desired esters such as isoamyl acetate and isobutyl acetate,
generally only slightly increasing the ethyl acetate content, that displays
an unpleasant “solvent” character at higher concentrations (Sarens &
Swiegers, 2014), so proving to be a good methodology to bring new
contributions to the aromatic improvement of beers. Yeasts of the
genus Williopsis (= Cyberlindnera), otherwise, seems to produce high
concentrations of acetate esters such as isoamyl acetate, and could be
used to exploit this feature in the production of beer in which fruity
notes are a desirable sensory trait (Lee, Ong, Yu, Curran, & Liu, 2010;
Li, Yu, Curran, & Liu, 2012).
Intense glucosidase activity is another interesting aspect in the
search for bioflavoring. Some Dekkera/Brettanomyces strains present
those enzymes, but W. anomalus has also an innate ability to markedly
produce extracellular β-glucosidase in media containing cellobiose. It
was shown that such enzymatic activity is enhanced in presence of eth-
anol, and inhibited by glucose, fructose and sucrose, a suggestive trait
for a final-stage inoculation as well as for bottle conditioning
(Swangkeaw, Vichitphan, Butzke, & Vichitphan, 2009).
One such promising example in this context is the use of starter co-
cultures including T. delbrueckii. In these conditions, it is possible to ver-
ify increased use of the yeast assimilable nitrogen (YAN) in the medium,
correlated to more expressive levels of some compounds – namely phe-
nyl ethyl acetate, ethyl hexanoate and ethyl octanoate – that can posi-
tively stamp complex fruity, floral and aniseed character to beers
(Canonico et al., 2016).
3.3. Low calorie beers
Beers with a low calorie content have achieved greater interest due
to obesity problems, especially in Western populations, which have
accounted for a growing market segment (Yeo & Liu, 2014). Low calorie
118 R.F. Basso et al. / Food Research International 86 (2016) 112–120
beers can be brewed addressing some alternative production methods,
particularly seeking to reduce the concentration of carbohydrates in the
final product. This type of beer may be done by both (a) special mashing
and collection of wort with great amount of fermentable sugars, or (b)
fermentative process of an ordinary wort under specific conditions
(Matthews, Byrne, & Hennigan, 2001).
A feasible method is the “dilution” of wort carbohydrates, which un-
fortunately leads to poorly structured beers, without much flavor ex-
pressiveness (Sato, Mizuno, Mukai, & Amano, 2006). Another possible
approach is the higher conversion of wort sugars during mashing,
aiming higher attenuation level in fermentation, thus lowering the
sugar content in the final product (Matthews et al., 2001). However,
with more fermentable sugars in the wort, more alcohol will be released
to the medium. Since ethanol has a higher energy value in comparison
to carbohydrates, the production of low calorie beers will not be possi-
ble unless part of the alcohol is later removed (Yeo & Liu, 2014). It is pos-
sible to drive fermentations by inoculating microorganisms with ability
to hydrolyze and assimilate more complex sugars, thus reducing the
concentration of residual sugars in the beer – as is the case of
Brettanomyces/Dekkera, that lead to low-calorie, slightly more alcoholic
beers (Steensels et al., 2015). Another possibility would be to increase
the concentration of those carbohydrates which are not assimilated
nor by yeasts, or by the human body, as is the case of β-glucans and
arabinoxylans (Yeo & Liu, 2014).
3.4. Low alcohol beers
An approach that is becoming more popular in brewing is the pro-
duction of beers with reduced alcohol content, considering two aspects
that deserve attention: health and physical integrity of consumers.
Methods to obtain beers with reduced alcohol content may be both
physical and biological. In the first case, it may be done by thermal tech-
niques (rectification; evaporation) or membrane usage (dialysis; re-
verse osmosis). Methods employing biological factors may rely on
traditional brewery plant (limited fermentation; changed mashing;
special yeasts), or in the use of specific machinery (continuous fermen-
tation) (Brányik, Silva, Baszczyňski, Lehnert, & Almeida e Silva, 2012).
Hence, new methods have been studied with the aim of producing
beverages with lower alcohol content, but keeping a superior sensory
quality. In this sense, non-Saccharomyces yeasts represent a very attrac-
tive alternative to produce low alcohol beers, since this technique does
not entail processing extra costs. In addition, it has the advantage of
avoiding the involuntary extraction of flavor compounds, a common
problem when ethanol extraction is carried out after fermentation by
distillation, reverse osmosis or dialysis (Erten & Campbell, 2001).
Wickerhamomyces subpelliculosus (= Pichia subpelliculosa) and
Cyberlindnera saturnus (=Williopsis saturnus), for example, showed in-
teresting results in the production of low alcohol wines with acceptable
flavor profiles (Erten & Campbell, 2001). Those latter abilities are also
related to alcohol acetyltransferase and esterase enzymes that enhance
production of aromatic isoamyl acetate and ethyl acetate (Erten &
Tanguler, 2010; Lee et al., 2010; Plata, Millán, Mauricio, & Ortega,
2003). The use of those yeasts in winemaking was possible by agitation
and aeration of the must during alcoholic fermentation suggesting its
application in brewing, even in mixed or sequential inoculation with
Saccharomyces.
Many strains of T. delbrueckii are only able to metabolize glucose,
fructose, and sucrose, so that they cannot degrade maltose, maltotriose,
and other complex carbohydrates. It should be an interesting way, once
the resulting beer would be much lower in alcohol content, usually close
to 0.9% (v/v), with the advantage that many of those strains impart rich
fruity flavor and aroma to the beer (Michel et al., 2016). Undoubtedly,
the use of T. delbrueckii represents a hopeful approach in this context,
once pure cultures of this species seem to reduce up to 50% the alcoholic
content, and simultaneously contribute to a complex fruity character in
the final beer (Canonico et al., 2016).
In this context, it is also possible to consider the use of cold contact
process in restrained fermentations, a methodology that allies very
low temperatures (~0 °C) to long fermentation period. Selected cold-re-
sistant strains are obviously necessary, and basal metabolism lead to
ester, higher alcohol, and carbonyl reduction, but virtually very little
ethanol production in low-density worts. Despite obtaining low/free-al-
cohol beers, this method enables to obtain a high volatile profile, and
low aldehyde reduction (Brányik et al., 2012; Perpète & Collin, 1999,
2000).
3.5. Functional beers
Functional beers – with health benefits for those who consume them
moderately – point to the use of non-conventional yeasts due to the po-
tential these microorganisms may play in production or transformation
of some beneficial compounds. The concept of functional beer is neces-
sarily combined to a low alcohol product, and to a matrix with substan-
tial amounts of fibers, vitamins, minerals and polyphenols (Yeo & Liu,
2014).
This is the case of melatonin is the sleep regulating hormone in
mammals and is a molecule that shows antioxidant properties. It is pro-
duced in ever-smaller amounts with human increasing age, and in such
case, it can be produced in beer during alcoholic fermentation by appro-
priate yeasts, constituting an exogenous source of this hormone, so ag-
gregating antioxidant, anti-aging, anti-inflammatory, antitumor and
immunomodulatory properties to the beer (Maldonado, Moreno, &
Calvo, 2009; Rodriguez-Naranjo, Gil-Izquierdo, Troncoso, Cantos-Villar,
& Garcia-Parrilla, 2011). Besides having the aforesaid advantages,
beers that undergo natural carbonation also present better nutritional
properties (García-Moreno, Calvo, & Maldonado, 2013). To date, how-
ever, no specific study attempted to relate fermentations driven by
non-Saccharomyces yeasts with the production of functional beers,
pointing the opportunity for future researches.
4. Conclusions
Many yeast species are emerging as candidates for the production of
specialty beers. Innovation not only relies on obtaining new products
with more complex aromatic and flavor character, but also on increas-
ing the range of approaches to achieve different results in a growing
market. Using non-Saccharomyces yeasts for pitching, in sequential or
co-inoculation, in bottle conditioning, or many other strategies, has pre-
sented the possibility to unveil old and new methods to produce spe-
cialty and original beers. Brewers are now reinterpreting the role of
different yeast species – regarded as spoilage microorganisms in uncon-
trolled or without good manufacturing practices – and learning with
them to find a multitude of applications and benefits in addition to
raw material. Yeasts from the genera Dekkera, Hanseniaspora, Pichia,
Torulaspora, Wickerhamomyces, and others, can offer a diversified enzy-
matic apparatus and bioconversion abilities that are allowing brewers
to work with new concepts that include bioflavoring, likewise beers
with reduced calorie and alcohol contents, or even functional beers.
Nonetheless, the introduction of a new yeast must be previously calcu-
lated, because each microorganism is unique, and can develop a range of
adaptations in contact with different substrates or conditions. Finally, it
is important not to ignore the importance of recent craft brewery revo-
lutions occurring in many parts of the globe, a movement that has been
paving the way to explore such traits and possibilities in order to find a
growing market of enthusiast and expert consumers.
Acknowledgements
This work was supported by Sao Paulo Research Foundation
(FAPESP) [grant number 2013/03766-4].
 R.F. Basso et al. / Food Research International 86 (2016) 112–120
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