Escolar Documentos
Profissional Documentos
Cultura Documentos
Could non-Saccharomyces yeasts contribute on innovative brewing fermenta-
tions?
PII: S0963-9969(16)30233-2
DOI: doi: 10.1016/j.foodres.2016.06.002
Reference: FRIN 6297
Please cite this article as: Basso, R.F., Alcarde, A.R. & Portugal, C.B., Could non-
Saccharomyces yeasts contribute on innovative brewing fermentations?, Food Research
International (2016), doi: 10.1016/j.foodres.2016.06.002
This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.
ACCEPTED MANUSCRIPT
T
a
Dept. Agroindústria, Alimentos e Nutrição, Escola Superior de Agricultura “Luiz de Queiroz” -
P
University of São Paulo, Avenida Pádua Dias 11, 13418-900, Piracicaba, Brazil.
RI
SC
Corresponding author: Cauré Barbosa Portugal
NU
Abstract
MA
With the advances in the production of beer worldwide, more challenges arise each year in the search
for new approaches to the development of distinctive beverages. Attempts to obtain products with
ED
more complex sensory characteristics have led experts and brewers to prospect for non-conventional
PT
yeasts, i.e., non-Saccharomyces yeasts that may provide a new range of perspectives in terms of
techniques and approaches. Besides the widespread use of Dekkera/Brettanomyces for the production
CE
of sour beers, other species are emerging for presenting unusual metabolic features that include the
AC
production of fruity esters, and a distinctive enzymatic apparatus. Wickerhamomyces anomalus and
Torulaspora delbrueckii stand out as the most promising yeasts in brewing processes. Such new
tendencies in the use of non-Saccharomyces yeasts comprise the production of low-alcohol beers,
functional beers, and bioflavoring approaches. This is a little explored field in brewing practice, still
requiring extensive research with practical application. In this sense, this review aims to present the
main points for the application of non-conventional yeasts in beer production, and thus contribute to
bioflavoring
ACCEPTED MANUSCRIPT
Chemical compounds
Ethanol (PubChem CID: 702); Acetic acid (PubChem CID: 176); Glucose (PubChem CID: 5793);
T
Maltose (PubChem CID: 6255); Maltotriose (PubChem CID: 192826); Ethyl acetate (PubChem CID:
P
8857); Isoamyl acetate (PubChem CID: 31276); Ethylguaiacol (PubChem CID: 62465); Ethylphenol
RI
(PubChem CID: 31242)
SC
Acknowledgements: This work was supported by Sao Paulo Research Foundation (FAPESP) [grant
number 2013/03766-4].
NU
MA
ED
PT
CE
AC
ACCEPTED MANUSCRIPT
1. Introduction
The brewing of beer-like beverages already occurred between 5500-3100 BC in the regions of
Mesopotamia and Ancient Egypt (Hornsey, 2007). Barley grew in wild forms in these regions, and
T
such civilizations found that moistening, germinating, and drying the grains would lead to a sweeter,
P
less perishable product (Papazian, 2003). This is what we now perceive as barley malt.
RI
Archaeological evidences suggest that in such times beer was made from barley, wheat, or a mixture
SC
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 malt and water, with further exposure to ambient
NU
air to induce “contamination” (Sicard & Legras, 2011).
MA
Since the species of the genus Saccharomyces – especially S. cerevisiae – frequently appeared as
abundant and dominant agents in spontaneous fermentations, they were intuitively selected over
generations, in different cultures and from distinct raw materials. Readily, men figured out that using
ED
a small piece or volume of a modified product into a fresh one, it worked as a starter for inducing
PT
fermentation again. It was only in the mid 17th century scientists began to understand what was
behind fermentation, enabling the isolation techniques, selection and use of pure inocula in those
CE
processes (Steensels & Verstrepen, 2014). Successive investigations, that included yeast’s
AC
description by Antonie van Leeuwenhoek in 1680, its association with beer wort fermentation by
Cagniard de Latour in 1836, the establishment of isolation techniques, and the use of pure starters as
top or bottom fermenting yeasts by Emil Hansen in 1883, enabled the improvement of controlled and
There are many different styles of beer but, primarily, the main brewing classification criterion
particularly relies on the type of fermentation. Although this concept may vary, brewing yeast are
or lager yeasts (Saccharomyces pastorianus). The two main styles of beer are still based on such
traits, of which top-fermenting yeasts display more genetic divergence, and the process normally
ACCEPTED MANUSCRIPT
occurs between 18 ºC – 24 ºC, while bottom-fermenting yeasts show a conserved genetic material,
and often ferment at lower temperatures (8 ° C – 14 ° C) (Lodolo et al., 2008). Fermentations for
T
microorganisms that are introduced by exposing the wort in shallow tanks during the overnight
P
cooling, before transferring it to wooden barrels for fermentation and aging.
RI
Since the establishment of sedentary life by mankind, and formation of the first human social
SC
structures, multiple events – including natural and human selection – led to a pool of domesticated or
selected yeasts (Sicard & Legras, 2011). Selected Saccharomyces strains are used with various
NU
purposes because of their plasticity on assimilating different substrates, usually not incorporated by
MA
S. cerevisiae. The use of Saccharomyces strains in controlled fermentations over decades is
essentially based on three main features: (a) efficient production of high ethanol amounts; (b) the use
of fermentation as the preferential metabolic pathway, combined to the positive Crabtree effect
ED
(repression of respiration by glucose); and (c) higher tolerance to ethanol and other environmental
PT
Nonetheless, with increasing demands for innovative products, other non-Saccharomyces species
CE
have been isolated and characterized for the development of specialty beers, with distinctive
AC
aromatic and flavor components (Vanderhaegen et al., 2003). They generally present low
fermentation yields, and are more sensitive to ethanol stress (Di Maro, Ercolini, & Coppola, 2007),
but may display a great range of possibilities for beer fermentations in order to provide distinctive
aroma and flavor (Renouf & Lonvaud-Funel, 2007). It has been shown, for example, that Dekkera
bruxellensis and Hanseniaspora uvarum (anamorph, Kloeckera apiculata) have the ability to
produce many esters with fruity character, besides promoting the enzymatic breakdown of some
proteins (De Keersmaecker, 1996; Kumara & Verachtert, 1991). Lately, other wild yeast species
have been mentioned as interesting alternatives in brewing by their genome particularities, as in the
microorganisms can display unusual metabolic abilities, thus changing the sensory character of the
final product by adding complexity in flavor, and modifying mouthfeel sensation (Spitaels et al.,
T
2014). Some yeasts are eventually characterized as spoilage agents, as in the case of
P
Dekkera/Brettanomyces and Debaryomyces – especially in poorly controlled processes in which they
RI
unintentionally appear. However, many of them are already used as pure inoculum, or in yeast blends
SC
under controlled conditions of processing for specialty beer production (Hayashi, Arai, Tada,
NU
In most beers, the microorganisms that carry out fermentation belong to different Saccharomyces
MA
species and strains (Bokulich & Bamforth, 2013). The occurrence of other species is commonly
reported in some peculiar beer styles produced by spontaneous fermentations, as the Belgian
Lambics and Gueuzes, and their American counterparts, Coolship Ales. However, due to the
ED
diversity of substrate assimilation patterns that non-conventional yeasts may display (van Dijken,
PT
2002), the use of non-Saccharomyces in controlled fermentations has been gaining popularity among
brewers in order to obtain distinctive products, a key point to gain market share in this growing
CE
segment (Ciani & Comitini, 2011; Cordero-Bueso, Esteve-Zarzoso, Cabellos, Gil-Díaz, & Arroyo,
AC
2013; González, Quirós, & Morales, 2013; Johnson, 2013). To take one example, the market of craft
beers in the United States grew 12.8 % in 2015, increasing 16.3 % in exports, and with incomes of
22.3 billion dollars, opening 620 craft breweries – only 68 closings in the same period – and
Kell, 2016). A significant strengthening of the sector has also been detected in several European
countries, where growth sometimes outstripped 300-1000% from 2009 to 2014 (Table 1).
The production of quality beer relies on the activity of fermenting yeasts that are qualified not only
for good fermentation yield-efficiency, but also for printing aroma and flavor to the beverage. Their
importance in beer production goes beyond the formation of ethanol and carbon dioxide, since most
ACCEPTED MANUSCRIPT
of the compounds that provide taste and aroma arise during fermentation, and these are intermediate
compounds and by-products of metabolism (Pires, Teixeira, Brányik, & Vicente, 2014).
In this sense, this review aims to recover and discuss recent information on the status of non-
T
conventional yeasts, and their potential application in beer production. Even so, this work intends to
P
present the main technological approaches regarding the use of non-Saccharomyces as a way to
RI
improve sensory traits, as well as product diversification and innovation in brewing processes.
SC
2. Non-Saccharomyces yeasts in brewing
NU
Saccharomyces cerevisiae is the widespread brewing yeast, and selected strains have proportionated
improvement and control of processes as a way to obtain chemical and sensory quality, as well as to
MA
maintain reproducibility. The expansion in the sector, and the growing number of specialized
consumers, have driven brewers to innovate methods changing hop varieties, using special malts,
ED
preparing distinctive blends, and more. Nevertheless, it is in the soul’s beverage, in the fermentation
PT
and pitching non-conventional yeasts, where producers are finding not only a multiplicity of aroma
and flavor production, but also new approaches and impressions in the overall organoleptic profile of
CE
beers (Michel et al., 2016). In this sense, some old acquaintances and other promissory
AC
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-conventional yeasts.
ACCEPTED MANUSCRIPT
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
T
production of sour beers, mainly by the discovery of its singular influence in Lambic beers, and have
P
attracted growing interest from brewers and connoisseurs. Lambics are produced under a complex
RI
succession of spontaneous fermentations, in which Dekkera/Brettanomyces yeasts take part during
SC
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
NU
that these yeasts are responsible for producing a set of flavor compounds, mainly ethyl phenol, ethyl
MA
guaiacol, isovaleric acid, acetic acid, and ethyl acetate, which together result in a typical flavor
character (Vanderhaegen et al., 2003; Verachtert & Derdelinckx, 2014). However, an increasing
number of researches addressing to the use of these yeasts in controlled fermentations has been
ED
observed in the last years, both in pure cultures as in co-inoculation with S. cerevisiae (Steensels &
PT
Verstrepen, 2014).
Beyond their typical phenolic notes, those commercial Dekkera/Brettanomyces strains have shown
CE
the ability to exert a marked influence on the sensory profile, contributing to an overall fruity or
AC
floral character by producing high levels of ethyl esters (ethyl acetate, ethyl caprate, ethyl caprylate,
ethyl lactate) (Crauwels et al., 2015). It is noteworthy that some peculiar flavors – namely “horse
blanket”, “stable” character – may also arise, and in small concentration, account for a positive and
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 amylases, although it a strain-dependent
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 or teleomorph form, and Brettanomyces designates the
T
anamorphic or asexual form of this yeast (Kurtzman, 2011). D. bruxellensis and D. anomala are the
P
species that typically appear in brewing processes (Steensels et al., 2015; White & Zainasheff, 2010).
RI
Besides presenting certain characteristics that enable their good performance in alcoholic
SC
fermentations, including tolerance to osmotic pressure, ethanol, limiting oxygen and low pH, these
species show a diverse metabolism that leads to interesting and unusual results in beer production
NU
(Steensels et al., 2015).
MA
When compared to S. cerevisiae, these yeasts have greater genetic diversity, and may present higher
gene doses due to increased ploidy variability, especially in low-oxygen and high sugar
concentration environments (Piškur et al., 2012; Woolfit, Rozpędowska, Piškur, & Wolfe, 2007). D.
ED
bruxellensis and D. anomala usually display the Crabtree effect, promoting fermentation instead of
PT
respiration, even with available oxygen in media with a certain concentration of sugars (Thomson et
al., 2005). However, the metabolic trait that markedly differentiates them from those traditional
CE
yeasts is the expression of the Custer effect. In this case, these organisms have greater ability to
AC
conduct the fermentation in the presence of oxygen by reducing or ceasing the glucose metabolism
(lag phase) under anaerobic conditions (Barnett & Entian, 2005). It is worth mentioning that small
oxygen inputs during fermentation is encouraging to best results in biomass production, fermentation
efficiency, total consumption of glucose, ethanol production, and low production of acetic acid
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 assimilate dextrin and starch (Steensels
et al., 2015). Cellobiose is a disaccharide found in wooden barrels used to mature beers (Daenen,
ACCEPTED MANUSCRIPT
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,
T
commonly produced by Dekkera/Brettanomyces, enables degradation of those complex molecules
P
into simple sugars that can be readily assimilated, resulting in a "super-attenuated" beer. The
RI
aforementioned methodology includes some particular traits, and practical results in beer styles in
SC
which low calorie and residual sugar content constitute innovative hot spots (Kumara, De Cort, &
NU
Regarding the influence of temperature on fermentations driven by Dekkera/Brettanomyces, most
MA
studies focus on biomass production rate, primary products (ethanol, acetic acid and carbon dioxide),
ethanol production, as well as lower acetic acid contents released in the medium (Brandam, Castro-
ED
Martínez, Délia, Ramón-Portugal, & Strehaiano, 2007). High concentration of these compounds can
PT
directly affect cell viability, aptitude in bioconversion of substrates, and low fermentative capacity.
Besides disturbing yeast growth and leading to acidic shock conditions, intense release of acetic acid
CE
also increases the risk of impairing the volatile acidity and contribute to sensory imbalance in the
AC
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 response of these microorganisms to ethanol stress entails on the extension 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,
The production of acetic acid by Dekkera/Brettanomyces is part of the strategy based on “make-
competitive edge, characterized by the production and accumulation of ethanol and acetic acid in the
T
fermentative environment, with subsequent degradation of these compounds when fermentable
P
sugars are depleted (Rozpędowska et al., 2011). Although considered a fault in many beer styles, low
RI
concentrations of acetic acid (< 1.2 g/l) are acceptable in those ones that use Dekkera/Brettanomyces
SC
in their primary fermentation (Steensels et al., 2015). Higher concentrations of this compound,
however, appear to inhibit substrate consumption. The metabolic regulation of this by-product is
NU
highly dependent on oxygen availability, with a direct relation between oxygen concentration in the
MA
substrate and the acetic acid production (Aguilar-Uscanga et al., 2003). It is known, moreover, that
the temperature 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
ED
beverage, mainly by the production of phenolic off-flavors (POF) that are frequently described as
PT
“rubber”, “burnt plastic”, “medicinal”, “leather”, and “barnyard”. Much of this influence relies on
the ability to metabolize hydroxycinnamic acids, of which ferulic, p-coumaric and caffeic acids are
CE
the most common (Chatonnet, Dubourdie, Boidron, & Pons, 1992; Lentz & Harris, 2015). These
AC
compounds are especially present in the bark, pericarp, aleurone, and endosperm of cereal grains,
associated to the arabinoxylans structural carbohydrates, and incorporated to wort during the
mashing process (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 respective 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;
ACCEPTED MANUSCRIPT
however, they are essential in many others, including the Belgian Lambic, Gueuze and Red Flanders,
the German Weizen and Rauchbier, the American Coolship Ale, as well as the Sour and Farmhouse
T
Primary fermentations using Dekkera/Brettanomyces generally lead to beers with low content of
P
isoamyl acetate and ethyl phenylethyl, but instead, high concentrations of ethyl acetate and ethyl
RI
lactate (Spaepen, Van Oevelen, & Verachtert, 1978; Spaepen & Verachtert, 1982). Moderate levels
SC
of these latter compounds are desirable, and may contribute to the flavor complexity of beers, but
higher concentrations may easily impart undesirable solvent-like, buttery and medicinal notes to the
NU
beverage (Cortés-Diéguez, Rodriguez-Solana, Domínguez, & Díaz, 2015). Ester imbalance occurs
MA
with the degradation of acetate esters by the esterase enzyme, a bioprocess much more efficient than
responsible for the hydrolysis of glycosides. Glycosides are non-volatile compounds composed by
PT
one aromatic molecule (aglycone) and one β-D-glucose (glycone), with possible additional sugar
units attached (Winterhalter & Skouroumounis, 1997). Such compounds are commonly found in
CE
plants, flowers and fruits, and some of them are used in brewing, and are present in hops and in the
AC
wood of barrels employed for maturation (Daenen, Saison, et al., 2008). The enzymatic hydrolysis of
glycosides releases the sugar molecules, further metabolized, and the aglycone, which can express
aromatic activity, representing a poorly explored methodology to improve the aromatic potential of
feature, but D. bruxellensis stands out for presenting such hydrolytic action on a number of
substrates, emerging as a good option for fermentations in pure culture or in co-culture with S.
other genera and species are reported to occur in brewing processes. Wickerhamomyces anomalus
T
and Torulaspora delbrueckii currently stand out as the most relevant non-conventional yeasts for
P
brewing due to their flavoring features, tolerance to extreme environmental conditions, low
RI
production of undesirable by-products, and frequent association with food and beverages (Canonico,
SC
Agarbati, Comitini, & Ciani, 2016; Michel et al., 2016; Schneider et al., 2012). Nonetheless, other
NU
usually occurring and isolated from spontaneous fermentations, have also been investigated. The
MA
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
anomala) is able to regulate the central metabolism of carbon according to oxygen availability
CE
pathway under oxygen restriction (Pasteur effect). It enables 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 assimilation, being able to properly metabolize hexoses (glucose, galactose, fructose), pentoses
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 ºC and 37 ºC), and under extreme pH values between 2.0 – 12.4 (Fredlund, 2004). Strains
ACCEPTED MANUSCRIPT
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).
T
Regarding its contribution to sensory active compounds, this species is a good producer of ethyl
P
propanoate, phenyl ethanol, 2-phenylethyl acetate, and especially ethyl acetate (Passoth et al., 2006).
RI
In contrast to S. cerevisiae, that synthesizes ethyl acetate mainly from acetyl-CoA and ethanol by the
SC
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,
NU
1981). Such compound is metabolically important because of its antifungal activity (Fredlund,
MA
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,
ED
Despite some evidences suggest an apparent inability to metabolize maltose (Walker, 2011), other
CE
studies show that some wild W. anomalus strains are indeed able to use this sugar and to grow even
AC
better than other commercial brewing yeasts (Y.-J. Lee et al., 2011). W. anomalus stands as a good
candidate in sequential or co-inoculated fermentations with other yeasts, always considering its
inhibitory action against some microorganisms (Fredlund et al., 2004; Passoth et al., 2006; Walker,
2011).
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 fermentation of Bavarian wheat beers (Michel
ACCEPTED MANUSCRIPT
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
T
curiously require oxygen to ferment – although not displaying the Custer effect (Alves-Araújo et al.,
P
2007; Hanl, Sommer, & Arneborg, 2004; Salvadó et al., 2011).
RI
T. delbrueckii shows invertase activity, necessary for sucrose hydrolysis, and subsequent
SC
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
NU
this species, highlighting that such biochemical apparatus can be inhibited by high concentrations of
MA
glucose in the medium. Interestingly, 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 unable to consume maltose and maltotriose (Alves-Araújo et
ED
Ethanol tolerance and fermentation efficiency are also strain-dependent features. Although it has
conditions (Tataridis et al., 2013), recent evidences demonstrate that many strains are only able to
AC
grow in concentrate beer wort with at most 5 % ethanol in the medium (Michel et al., 2016).
Tolerance to iso-α-acids from hops is another important trait that yeast should display in brewing
conditions, once that compound has inhibitory 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 iso-α-acids in the medium, a concentration that correlates to highly hopped
beer styles. Resistance to such compounds derived from hops can be evident even in the 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 iso-α-acid (Michel et al., 2016). Of course, these are strain-
dependent traits, but the ability to deal with some ethanol content in the medium – toxic to the cell –
ACCEPTED MANUSCRIPT
provides better membrane selectivity conditions. As a result, some strains of T. delbrueckii are less
sensitive to antimicrobial effects of iso-α-acids, and may be proposed as a tool for average strength
T
The most interesting contribution of this yeast is perhaps the profile of volatile compounds it may
P
confer to the final beer. Some recent investigations reveal the ability of some strains to produce 2-
RI
phenylethanol (sweetish, “rose petals” and floral flavors) (Etschmann et al., 2015), n-propanol and i-
SC
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.,
NU
2008), as in the case of ethyl hexanoate, ethyl octanoate and ethyl decanoate, whose concentrations
MA
were negligible, and below the perception threshold (0.01 mg/l) (Michel et al., 2016). Unlike
not present in T. delbrueckiii, so that this species does not have the inconvenience of producing
ED
phenolic off-flavors. The same is not true in the case of diacetyl production, since it often releases
PT
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
CE
The specialty beer market has gained attention worldwide in the last 30 years. In addition to the
evidence of beers produced with better quality inputs, and using methods that respect the final
product attributes, the “liquid bread” is gaining other perspectives. These new insights include 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 extracellular enzymes, besides singular fermentative ability or sugar assimilation traits,
ACCEPTED MANUSCRIPT
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 enhance flavor
T
characteristics, to obtain products with low calorie and/or alcohol contents, and even being able to
P
provide beers with current complexions of a functional food.
RI
3.1. Recovering old production methods for innovation
SC
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
NU
popularity of pure yeast cultures and stainless steel vats. Today, this procedure has been gaining
MA
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
ED
harbor some wild yeasts, lactic acid bacteria, and acetic acid bacteria (Bokulich, Bamforth, & Mills,
PT
2012; Verachtert & Derdelinckx, 2014). These microorganisms can play a positive role on
characteristics of the product, as is the case of sour beers, where some specific metabolic traits will
CE
impart very particular complexity in flavor, aroma and acidity (Spitaels et al., 2014; Tonsmeire,
AC
Previously, bottle conditioning was the only choice for carbonating beers until the advent of
fermentation and maturation 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 addition to eliminating dissolved oxygen that could
contribute to precipitated deterioration of beer (Vanderhaegen et al., 2003). This is the case of
Gueuze beers, wherein young Lambics (up to one year maturation) confer complex sugars to the
mixture, and old Lambics (two years or more) “inoculate” new microorganisms – especially
ACCEPTED MANUSCRIPT
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,
T
optionally, pure cultures of different yeast strains to increase the complexity of final product
P
(Vanderhaegen et al., 2003).
RI
3.2. Bioflavoring
SC
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
NU
bioflavoring comes up for the synthesis and transformation of flavor compounds by biological
MA
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
ED
specific yeast species; (b) sequential or co-inoculation with different microorganisms; (c) use of
PT
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
CE
The sequential inoculation of Pichia kluyveri in beer wort is an example, where the combined effect
of different hop varieties showed to improve 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
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 (P.-R. Lee, Ong, Yu, Curran, & Liu, 2010; Li, Yu,
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
T
enzymatic activity is enhanced in presence of ethanol, and inhibited by glucose, fructose and sucrose,
P
a suggestive trait for a final-stage inoculation as well as for bottle conditioning (Swangkeaw,
RI
Vichitphan, Butzke, & Vichitphan, 2009).
SC
One such promising example in this context is the use of starter co-cultures including T. delbrueckii.
In these conditions, it is possible to verify increased use of the yeast assimilable nitrogen (YAN) in
NU
the medium, correlated to more expressive levels of some compounds – namely phenyl ethyl acetate,
MA
ethyl hexanoate and ethyl octanoate – that can positively stamp complex fruity, floral and aniseed
Beers with a low calorie content have achieved greater interest due to obesity problems, especially in
PT
Western populations, which have accounted for a growing market segment (Yeo & Liu, 2014). Low
calorie beers can be brewed addressing some alternative production methods, particularly seeking to
CE
reduce the concentration of carbohydrates in the final product. This type of beer may be done by both
AC
(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 unfortunately leads to poorly
structured beers, without much flavor expressiveness (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
ACCEPTED MANUSCRIPT
low calorie beers will not be possible unless part of the alcohol is later removed (Yeo & Liu, 2014).
assimilate more complex sugars, thus reducing the concentration of residual sugars in the beer – as is
T
the case of Brettanomyces/Dekkera, that lead to low-calorie, slightly more alcoholic beers (Steensels
P
et al., 2015). Another possibility would be to increase the concentration of those carbohydrates
RI
which are not assimilated nor by yeasts, or by the human body, as is the case of β-glucans and
SC
arabinoxylans (Yeo & Liu, 2014).
NU
An approach that is becoming more popular in brewing is the production of beers with reduced
MA
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 techniques (rectification; evaporation) or
ED
membrane usage (dialysis; reverse osmosis). Methods employing biological factors may rely on
PT
traditional brewery plant (limited fermentation; changed mashing; special yeasts), or in the use of
specific machinery (continuous fermentation) (Brányik, Silva, Baszczyňski, Lehnert, & Almeida e
CE
Silva, 2012).
AC
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 attractive 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
Williopsis saturnus), for example, showed interesting results in the production of low alcohol wines
with acceptable flavor profiles (Erten & Campbell, 2001),. Those latter abilities are also related to
ACCEPTED MANUSCRIPT
alcohol acetyltransferase and esterase enzymes that enhance production of aromatic isoamyl acetate
and ethyl acetate (Erten & Tanguler, 2010; P.-R. 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
T
during alcoholic fermentation suggesting its application in brewing, even in mixed or sequential
P
inoculation with Saccharomyces.
RI
Many strains of T. delbrueckii are only able to metabolize glucose, fructose, and sucrose, so that they
SC
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),
NU
with the advantage that many of those strains impart rich fruity flavor and aroma to the beer (Michel
MA
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).
ED
In this context, it is also possible to consider the use of cold contact process in restrained
PT
fermentations, a methodology that allies very low temperatures (~ 0 ºC) to long fermentation period.
Selected cold-resistant strains are obviously necessary, and basal metabolism lead to ester, higher
CE
alcohol, and carbonyl reduction, but virtually very little ethanol production in low-density worts.
AC
Despite obtaining low/free-alcohol beers, this method enables to obtain a high volatile profile, and
low aldehyde reduction (Brányik et al., 2012; Perpète & Collin, 1999, 2000).
Functional beers – with health benefits for those who consume them moderately – point to the use of
non-conventional yeasts due to the potential these microorganisms may play in production or
combined to a low alcohol product, and to a matrix with substantial amounts of fibers, vitamins,
This is the case of melatonin is the sleep regulating hormone in mammals and is a molecule that
shows antioxidant properties. It is produced in ever-smaller amounts with human increasing age, and
in such case, it can be produced in beer during alcoholic fermentation by appropriate yeasts,
T
constituting an exogenous source of this hormone, so aggregating antioxidant, anti-aging, anti-
P
inflammatory, antitumor and immunomodulatory properties to the beer (Maldonado, Moreno, &
RI
Calvo, 2009; Rodriguez-Naranjo, Gil-Izquierdo, Troncoso, Cantos-Villar, & Garcia-Parrilla, 2011).
SC
Besides having the aforesaid advantages, beers that undergo natural carbonation also present better
nutritional properties (García-Moreno, Calvo, & Maldonado, 2013). To date, however, no specific
NU
study attempted to relate fermentations driven by non-Saccharomyces yeasts with the production of
MA
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
ED
only relies on obtaining new products with more complex aromatic and flavor character, but also on
PT
increasing the range of approaches to achieve different results in a growing market. Using non-
other strategies, has presented the possibility to unveil old and new methods to produce specialty and
AC
original beers. Brewers are now reinterpreting the role of different yeast species – regarded as
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 enzymatic 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 calculated,
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
ACCEPTED MANUSCRIPT
brewery revolutions 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.
P T
RI
SC
NU
MA
ED
PT
CE
AC
ACCEPTED MANUSCRIPT
microbreweries relative
2009 2014 growth (%)
Spain 27 314 1062
Norway 13 65 400
T
Sweden 30 149 396
P
Czech Republic 51 238 366
RI
Slovakia 9 39 333
Slovenia 20 49 145
Italy 242 585 141
SC
France 263 566 115
United Kingdom 694 1414 103
Switzerland 232 440 89
NU
Adapted from: Beers Statistics 2015 (The Brewers of Europe)
MA
ED
PT
CE
AC
ACCEPTED MANUSCRIPT
Table 2 Taxonomical status, isolation sources and use of non-conventional yeasts for brewing
T
IP
species anamorph a synonyms a isolation sources b application food safety c
CR
Cyberlindnera saturnus Williopsis saturnus; insect, soil bioflavoring; low
Saccharomyces saturnus alcohol beer safe
S
Dekkera anomala Brettanomyces anomalus Dekkera/Brettanomyces beer, cider, soft drink bioflavoring; low
NU
safe
claussenii calorie beer
Dekkera bruxellensis Brettanomyces bruxellensis Brettanomyces lambicus; beer, grape, wine, bioflavoring; low
safe
MA
Brettanomyces custersii ginger ale calorie beer
Hanseniaspora uvarum Kloeckera apiculata Kloeckeraspora uvarum; grape must, fruits, soil, bioflavoring
Hanseniaspora apiculata sour dough, insect, safe
ED
flowers
Pichia kluyveri Hansenula kluyveri olives, fruits, flowers, bioflavoring
safe
soil
Torulaspora delbrueckii Candida colliculosa
PT Saccharomyces rosei;
Saccharomyces delbrueckii
beer, fruits, soil, milk,
moss, mushroom
bioflavoring; low
alcohol beer
safe
CE
Wickerhamomyces subpelliculosus Pichia subpelliculosa; fruits; vegetables; low alcohol beer
safe
Hansenula subpelliculosa molasses; jam; milk
AC
Wickerhamomyces anomalus Candida beverwijkiae Pichia anomala; beer, sour dough, fruits, bioflavoring; low
Saccharomyces anomalus; sake, wine, oak calorie beer safe
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)
ACCEPTED MANUSCRIPT
Table 3 Major aromatic compounds, enzymes and peculiar traits of non-conventional yeasts in brewing
T
aromatic prominent
species flavor compound disadvantage peculiar traits references
descriptor enzyme
IP
C. saturnus isoamyl acetate banana, pear AAT biofilm formation ethanol sensitive (Erten & Campbell, 2001; Erten &
CR
ethyl acetate fruity, solvent Tanguler, 2010; P.-R. Lee et al.,
2010)
D. anomala acetic acid vinegary BGL, CDC, high acetic acid osmotolerant; pH tolerant; (Lentz & Harris, 2015)
S
ehtyl phenols clove; leather VPR production; overall Custer effect
ethyl esters fruity off-flavor
NU
D. bruxellensis acetic acid vinegary BGL, CDC, high acetic acid osmotolerant; pH tolerant; (Lentz & Harris, 2015)
ehtyl phenols clove; leather VPR production; overall Custer effect
ethyl esters fruity off-flavor
MA
H. uvarum isoamyl acetate banana; pear BGL cycloheximide-resistant; (Arévalo Villena, Úbeda Iranzo,
ethyl acetate fruity; solvent ethanol tolerant Cordero Otero, & Briones Pérez,
2005; Spitaels et al., 2014; Wang,
Mas, & Esteve-Zarzoso, 2015)
ED
P. kluyveri isoamyl acetate banana; pear AAT osmotolerant; Pasteur (Fredlund, 2004; Ma, He, Cao, Bai,
isobutyl acetate banana; fruity effect; pH tolerant & Li, 2016; Sarens & Swiegers,
2014; Steensels & Verstrepen, 2014;
PT
Walker, 2011)
T. delbrueckii isoamyl acetate banana; pear AAT, EST, high acetaldehyde osmotolerant; (Canonico et al., 2016; Charoenchai,
ethyl butyrate banana; apple; INV, BGL production cryotolerant; ethanol Fleet, Henschke, & Todd, 1997;
CE
strawberry tolerant Peinado, Moreno, Bueno, Moreno,
acetaldehyde green apple & Mauricio, 2004; Plata et al., 2003)
W. subpelliculosus isoamyl acetate banana, pear AAT, EST biofilm formation; (Erten & Campbell, 2001; Plata et
AC
References
Aguilar-Uscanga, M. G., Délia, M. L., & Strehaiano, P. (2003). Brettanomyces bruxellensis: effect of
oxygen on growth and acetic acid production. Applied Microbiology and Biotechnology,
T
61(2), 157-162. doi: 10.1007/s00253-002-1197-z
P
Albertin, W., Chasseriaud, L., Comte, G., Panfili, A., Delcamp, A., Salin, F., . . . Bely, M. (2014).
RI
Winemaking and bioprocesses strongly shaped the genetic diversity of the ubiquitous yeast
SC
Torulaspora delbrueckii. PLoS ONE, 9(4), e94246. doi: 10.1371/journal.pone.0094246
Alves-Araújo, C., Pacheco, A., Almeida, M. J., Spencer-Martins, I., Leão, C., & Sousa, M. J. (2007).
NU
Sugar utilization patterns and respiro-fermentative metabolism in the baker's yeast
MA
Torulaspora delbrueckii. Microbiology, 153(3), 898-904. doi:
doi:10.1099/mic.0.2006/003475-0
Arévalo Villena, M., Úbeda Iranzo, J. F., Cordero Otero, R. R., & Briones Pérez, A. I. (2005).
ED
Optimization of a rapid method for studying the cellular location of β-glucosidase activity in
PT
2672.2005.02627.x
CE
Barata, A., Caldeira, J., Botelheiro, R., Pagliara, A., Malfeito-Ferreira, M., & Loureiro, V. (2008).
AC
Survival patterns of Dekkera bruxellensis in wines and inhibitory effect of sulphur dioxide.
Barnett, J. A., & Entian, K. D. (2005). A history of research on yeasts. 9: Regulation of sugar
Bely, M., Stoeckle, P., Masneuf-Pomarède, I., & Dubourdieu, D. (2008). Impact of mixed
http://dx.doi.org/10.1016/j.ijfoodmicro.2007.12.023
ACCEPTED MANUSCRIPT
Bokulich, N. A., & Bamforth, C. W. (2013). The microbiology of malting and brewing.
Bokulich, N. A., Bamforth, C. W., & Mills, D. A. (2012). Brewhouse-resident microbiota are
T
responsible for multi-stage fermentation of American Coolship Ale. PLoS ONE, 7(4),
P
e35507. doi: 10.1371/journal.pone.0035507
RI
Bourdichon, F., Casaregola, S., Farrokh, C., Frisvad, J. C., Gerds, M. L., Hammes, W. P., . . .
SC
Hansen, E. B. (2012). Food fermentations: Microorganisms with technological beneficial use.
NU
http://dx.doi.org/10.1016/j.ijfoodmicro.2011.12.030
MA
Brandam, C., Castro-Martínez, C., Délia, M.-L., Ramón-Portugal, F., & Strehaiano, P. (2007). Effect
Brányik, T., Silva, D. P., Baszczyňski, M., Lehnert, R., & Almeida e Silva, J. B. (2012). A review of
PT
methods of low alcohol and alcohol-free beer production. Journal of Food Engineering,
Brewers-Association. (2016). National beer sales and production data. The New Brewer Retrieved
AC
production-data/
Canonico, L., Agarbati, A., Comitini, F., & Ciani, M. (2016). Torulaspora delbrueckii in the brewing
process: A new approach to enhance bioflavour and to reduce ethanol content. Food
Charoenchai, C., Fleet, G. H., Henschke, P. A., & Todd, B. E. N. T. (1997). Screening of non-
Saccharomyces wine yeasts for the presence of extracellular hydrolytic enzymes. Australian
Chatonnet, P., Dubourdie, D., Boidron, J. N. l., & Pons, M. (1992). The origin of ethylphenols in
165-178.
T
Ciani, M., & Comitini, F. (2011). Non-Saccharomyces wine yeasts have a promising role in
P
biotechnological approaches to winemaking. Annals of Microbiology, 61(1), 25-32.
RI
Conterno, L., Aprea, E., Franceschi, P., Viola, R., & Vrhovsek, U. (2013). Overview of Dekkera
SC
bruxellensis behaviour in an ethanol-rich environment using untargeted and targeted
NU
10.1016/j.foodres.2013.01.049
MA
Cordero-Bueso, G., Esteve-Zarzoso, B., Cabellos, J., Gil-Díaz, M., & Arroyo, T. (2013).
fermentations of Malvar (Vitis vinifera cv. L.). European Food Research and Technology,
ED
Cortés-Diéguez, S., Rodriguez-Solana, R., Domínguez, J. M., & Díaz, E. (2015). Impact odorants
and sensory profile of young red wines from four Galician (NW of Spain) traditional
CE
Crauwels, S., Steensels, J., Aerts, G., Willems, K. A., Verstrepen, K. J., & Lievens, B. (2015).
68(September/October), 110-121.
Daenen, L., Saison, D., Sterckx, F., Delvaux, F. R., Verachtert, H., & Derdelinckx, G. (2008).
10.1111/j.1365-2672.2007.03566.x
ACCEPTED MANUSCRIPT
Daenen, L., Sterckx, F., Delvaux, F. R., Verachtert, H., & Derdelinckx, G. (2008). Evaluation of the
(Prunus cerasus L.) used in the production of special fruit beers. FEMS Yeast Research, 8(7),
T
1103-1114. doi: 10.1111/j.1567-1364.2008.00421.x
P
De Keersmaecker, J. (1996). The mystery of lambic beer. Scientific American 275, 74-81.
RI
Di Maro, E., Ercolini, D., & Coppola, S. (2007). Yeast dynamics during spontaneous wine
SC
fermentation of the Catalanesca grape. International Journal of Food Microbiology, 117(2),
NU
Erten, H., & Campbell, I. (2001). The production of low-alcohol wines by aerobic yeasts. Journal of
MA
the Institute of Brewing, 59(3), 207-215. doi: 10.1002/j.2050-0416.1953.tb06929.x
Erten, H., & Tanguler, H. (2010). Influence of Williopsis saturnus yeasts in combination with
Etschmann, M., Huth, I., Walisko, R., Schuster, J., Krull, R., Holtmann, D., . . . Schrader, J. (2015).
Fredlund, E. (2004). Central carbon metabolism of the biocontrol yeast Pichia anomala: Influence of
Uppsala. (488)
Fredlund, E., Druvefors, U. Ä., Olstorpe, M. N., Passoth, V., & Schnürer, J. (2004). Influence of
ethyl acetate production and ploidy on the anti-mould activity of Pichia anomala. FEMS
García-Moreno, H., Calvo, J. R., & Maldonado, M. D. (2013). High levels of melatonin generated
during the brewing process. Journal of Pineal Research, 55(1), 26-30. doi: 10.1111/jpi.12005
ACCEPTED MANUSCRIPT
Goldammer, T. (2008). The brewers' handbook. The complete book to brewing beer (2 ed.). Clifton:
Apex Publishers.
González, R., Quirós, M., & Morales, P. (2013). Yeast respiration of sugars by non-Saccharomyces
T
yeast species: A promising and barely explored approach to lowering alcohol content
P
of wines. Trends in Food Science & Technology, 29(1), 55-61. doi:
RI
10.1016/j.tifs.2012.06.015
SC
Hanl, L., Sommer, P., & Arneborg, N. (2004). The effect of decreasing oxygen feed rates on growth
NU
Biotechnology, 67(1), 113-118. doi: 10.1007/s00253-004-1695-2
MA
Harris, V., Ford, C., Jiranek, V., & Grbin, P. (2009). Survey of enzyme activity responsible for
Hayashi, N., Arai, R., Tada, S., Taguchi, H., & Ogawa, Y. (2007). Detection and identification of
PT
Hernández-Orte, P., Cersosimo, M., Loscos, N., Cacho, J., Garcia-Moruno, E., & Ferreira, V. (2008).
AC
The development of varietal aroma from non-floral grapes by yeasts of different genera. Food
Hornsey, I. (2007). The Chemistry and Biology of winemaking. Cambridge: The Royal Society of
Chemistry.
Kell, J. (2016). What You Didn't Know About the Boom in Craft Beer Retrieved 24/05/2016, 2016,
from http://fortune.com/2016/03/22/craft-beer-sales-rise-2015/
ACCEPTED MANUSCRIPT
Kumara, H. M. C. S., De Cort, S., & Verachtert, H. (1993). Localization and characterization of α-
59(8), 2352-2358.
T
Kumara, H. M. C. S., & Verachtert, H. (1991). Identification of lambic supperattenuating micro-
P
organisms by the use of selective antibiotics. Journal of the Institute of Brewing, 97(3), 181-
RI
185. doi: 10.1002/j.2050-0416.1991.tb01064.x
SC
Kurtzman, C. (2011). Phylogeny of the ascomycetous yeasts and the renaming of Pichia anomala to
NU
Lee, P.-R., Ong, Y.-L., Yu, B., Curran, P., & Liu, S.-Q. (2010). Evolution of volatile compounds in
MA
papaya wine fermented with three Williopsis saturnus yeasts. International Journal of Food
Lee, Y.-J., Choi, Y.-R., Lee, S.-Y., Park, J.-T., Shim, J.-H., Park, K.-H., & Kim, J.-W. (2011).
ED
Screening wild yeast strains for alcohol fermentation from various fruits. Mycobiology, 39(1),
PT
Lentz, M., & Harris, C. (2015). Analysis of growth inhibition and metabolism of hydroxycinnamic
CE
acids by brewing and spoilage strains of Brettanomyces yeast. Foods, 4(4), 581-593.
AC
Li, X., Yu, B., Curran, P., & Liu, S.-Q. (2012). Impact of two Williopsis yeast strains on the volatile
composition of mango wine. International Journal of Food Science & Technology, 47(4),
Lodolo, E. J., Kock, J. L. F., Axcell, B. C., & Brooks, M. (2008). The yeast Saccharomyces
cerevisiae – the main character in beer brewing. FEMS Yeast Research, 8(7), 1018-1036. doi:
10.1111/j.1567-1364.2008.00433.x
Ma, C., He, Y., Cao, Y., Bai, X., & Li, H. (2016). Analysis of flavour compounds in beer with
doi: 10.1002/jib.330
Maldonado, M. D., Moreno, H., & Calvo, J. R. (2009). Melatonin present in beer contributes to
T
increase the levels of melatonin and antioxidant capacity of the human serum. Clinical
P
Nutrition, 28(2), 188-191. doi: http://dx.doi.org/10.1016/j.clnu.2009.02.001
RI
Masneuf-Pomarede, I., Bely, M., Marullo, P., & Albertin, W. (2015). The genetics of non-
SC
conventional wine yeasts: current knowledge and future challenges. Frontiers in
NU
Matthews, S. L., Byrne, H., & Hennigan, G. P. (2001). Preparation of a low carbohydrate beer by
MA
mashing at high temperature with glucoamylase. Journal of the Institute of Brewing, 107(3),
Meilgaard, M. C. (1982). Prediction of flavor differences between beers from their chemical
ED
10.1021/jf00114a002
Michel, M., Kopecká, J., Meier-Dörnberg, T., Zarnkow, M., Jacob, F., & Hutzler, M. (2016).
CE
Screening for new brewing yeasts in the non-Saccharomyces sector with Torulaspora
AC
Papazian, C. (2003). The complete joy of homebrewing (3 ed.). New York: Harper Collins.
Passoth, V., Fredlund, E., Druvefors, U. Ä., & Schnürer, J. (2006). Biotechnology, physiology and
genetics of the yeast Pichia anomala. FEMS Yeast Research, 6(1), 3-13. doi: 10.1111/j.1567-
1364.2005.00004.x
Peinado, R. A., Moreno, J., Bueno, J. E., Moreno, J. A., & Mauricio, J. C. (2004). Comparative study
Perpète, P., & Collin, S. (1999). Fate of the worty flavours in a cold contact fermentation. Food
Perpète, P., & Collin, S. (2000). How to improve the enzymatic worty flavour reduction in a cold
T
contact fermentation. Food Chemistry, 70(4), 457-462. doi: http://dx.doi.org/10.1016/S0308-
P
8146(00)00111-4
RI
Pires, E., Teixeira, J., Brányik, T., & Vicente, A. (2014). Yeast: the soul of beer’s aroma - a review
SC
of flavour-active esters and higher alcohols produced by the brewing yeast. Applied
NU
Piškur, J., Ling, Z., Marcet-Houben, M., Ishchuk, O. P., Aerts, A., LaButti, K., . . . Phister, T. (2012).
MA
The genome of wine yeast Dekkera bruxellensis provides a tool to explore its food-related
http://dx.doi.org/10.1016/j.ijfoodmicro.2012.05.008
ED
Plata, C., Millán, C., Mauricio, J. C., & Ortega, J. M. (2003). Formation of ethyl acetate and isoamyl
PT
acetate by various species of wine yeasts. Food Microbiology, 20(2), 217-224. doi:
http://dx.doi.org/10.1016/S0740-0020(02)00101-6
CE
Portugal, C., Sáenz, Y., Rojo-Bezares, B., Zarazaga, M., Torres, C., Cacho, J., & Ruiz-Larrea, F.
AC
practical approaches. European Food Research and Technology, 238(4), 641-652. doi:
10.1007/s00217-013-2143-2
Rodriguez-Naranjo, M. I., Gil-Izquierdo, A., Troncoso, A. M., Cantos-Villar, E., & Garcia-Parrilla,
Rogers, C. M., Veatch, D., Covey, A., Staton, C., & Bochman, M. L. (2016). Terminal acidic shock
inhibits sour beer bottle conditioning by Saccharomyces cerevisiae. Food Microbiology, 57,
T
Rojas, V., Gil, J. V., Manzanares, P., Gavara, R., Piñaga, F., & Flors, A. (2002). Measurement of
P
alcohol acetyltransferase and ester hydrolase activities in yeast extracts. Enzyme and
RI
Microbial Technology, 30(2), 224-230. doi: 10.1016/S0141-0229(01)00483-5
SC
Rozpędowska, E., Hellborg, L., Ishchuk, O. P., Orhan, F., Galafassi, S., Merico, A., . . . Piskur, J.
NU
Dekkera yeasts. Nature Communications, 2(302), 1-7. doi: 10.1038/ncomms1305
MA
Salvadó, Z., Arroyo-López, F. N., Guillamón, J. M., Salazar, G., Querol, A., & Barrio, E. (2011).
Sarens, S., & Swiegers, J. H. (2014). United States Patent No. US20140234480A1.
PT
Sato, K., Mizuno, A., Mukai, N., & Amano, H. (2006). United States Patent No.: US7115289.
Schneider, J., Rupp, O., Trost, E., Jaenicke, S., Passoth, V., Goesmann, A., . . . Brinkrolf, K. (2012).
CE
doi: 10.1111/j.1567-1364.2012.00791.x
Sicard, D., & Legras, J.-L. (2011). Bread, beer and wine: Yeast domestication in the Saccharomyces
10.1016/j.crvi.2010.12.016
Spaepen, M., Van Oevelen, D., & Verachtert, H. (1978). Fatty acids and esters produced during the
spontaneous fermentation of Lambic and Gueuze. Journal of the Institute of Brewing, 84(5),
Spaepen, M., & Verachtert, H. (1982). Esterase activity in the genus Brettanomyces. Journal of the
Spitaels, F., Wieme, A. D., Janssens, M., Aerts, M., Daniel, H.-M., Van Landschoot, A., . . .
T
Vandamme, P. (2014). The microbial diversity of traditional spontaneously fermented lambic
P
beer. PLoS ONE, 9(4), 1-13. doi: 10.1371/journal.pone.0095384
RI
Steensels, J., Daenen, L., Malcorps, P., Derdelinckx, G., Verachtert, H., & Verstrepen, K. J. (2015).
SC
Brettanomyces yeasts - From spoilage organisms to valuable contributors to industrial
NU
10.1016/j.ijfoodmicro.2015.04.005
MA
Steensels, J., & Verstrepen, K. J. (2014). Taming wild yeast: potential of conventional and
Swangkeaw, J., Vichitphan, S., Butzke, C., & Vichitphan, K. (2009). The characterisation of a novel
PT
Tataridis, P., Kanelis, A., Logotetis, S., & Nerancis, E. (2013). Use of non-Saccharomyces
AC
Thomson, J. M., Gaucher, E. A., Burgan, M. F., De Kee, D. W., Li, T., Aris, J. P., & Benner, S. A.
(2005). Resurrecting ancestral alcohol dehydrogenases from yeast. Nature genetics, 37(6),
630-635.
Tonsmeire, M. (2014). American sour beer: innovative techniques for mixed fermentations. Boulder:
Brewers Publications.
van Dijken, H. (2002). Biochemistry, genetics, biotechnology and ecology of non-conventional yeasts
(NCY). Paper presented at the The 21st International Specialized Symposium on Yeasts Lviv
ACCEPTED MANUSCRIPT
https://femsyr.oxfordjournals.org/content/1/4/337
Vanbeneden, N., Gils, F., Delvaux, F., & Delvaux, F. R. (2008). Formation of 4-vinyl and 4-ethyl
T
derivatives from hydroxycinnamic acids: Occurrence of volatile phenolic flavour compounds
P
in beer and distribution of Pad1-activity among brewing yeasts. Food Chemistry, 107(1),
RI
221-230. doi: 10.1016/j.foodchem.2007.08.008
SC
Vanbeneden, N., Van Roey, T., Willems, F., Delvaux, F., & Delvaux, F. R. (2008). Release of
phenolic flavour precursors during wort production: Influence of process parameters and grist
NU
composition on ferulic acid release during brewing. Food Chemistry, 111(1), 83-91. doi:
10.1016/j.foodchem.2008.03.029
MA
Vanderhaegen, B., Neven, H., Coghe, S., Verstrepen, K. J., Derdelinckx, G., & Verachtert, H.
Verachtert, H., & Derdelinckx, G. (2014). Belgian acidic beers: daily reminiscences of the past.
Walker, G. (2011). Pichia anomala: cell physiology and biotechnology relative to other yeasts.
AC
Wang, C., Mas, A., & Esteve-Zarzoso, B. (2015). Interaction between Hanseniaspora uvarum and
White, C., & Zainasheff, J. (2010). Yeast: The practical guide to beer fermentation. Boulder:
Brewers Publications.
Villadsen, U. Stockar & C. Wandrey (Eds.), Biotechnology of Aroma Compounds (pp. 73-
T
Woolfit, M., Rozpędowska, E., Piškur, J., & Wolfe, K. H. (2007). Genome survey sequencing of the
P
wine spoilage yeast Dekkera (Brettanomyces) bruxellensis. Eukaryotic Cell, 6(4), 721-733.
RI
doi: 10.1128/ec.00338-06
SC
Yeo, H. Q., & Liu, S.-Q. (2014). An overview of selected specialty beers: developments, challenges
and prospects. International Journal of Food Science & Technology, 49(7), 1607-1618. doi:
NU
10.1111/ijfs.12488
MA
Yoshioka, K., & Hashimoto, N. (1981). Ester formation by alcohol acetyltransferase from brewers’
Yu, J., Vasanthan, T., & Temelli, F. (2001). Analysis of phenolic acids in barley by high-
ED
P T
RI
SC
NU
MA
Graphical Abstract
ED
PT
CE
Highlights