Production of sensory compounds by means of the yeast Dekkera bruxellensis ...

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Special Issue - Bioflavours

Production of sensory compounds by means of the yeast Dekkera bruxellensis in

different nitrogen sources with the prospect of producing cachaça

Denise Castro Parente1, Esteban Espinosa Vidal

5, Fernanda Cristina Bezerra Leite

1,4, Will de

Barros Pita1,3

and Marcos Antonio de Morais Jr.1,2,*

1Interdepartmental Research Group on Metabolic Engineering,

2Department of Genetics and

3Department of Antibiotics. Federal University of Pernambuco, Av. Moraes Rego 1235,

50670-901, Recife, PE, Brazil.

4Department of Biology. Federal Rural University of Pernambuco, Rua Dom Manoel de

Medeiros, s/n, 52171-900, Recife, PE, Brasil.

5Laboratory of Bioprocessing, Center for Strategic Technologies Northeast, Av. Luiz Freire,

01, 50740-540, Recife PE, Brazil.

*Corresponding author:

Marcos Antonio de Morais Jr.

Departamento de Genética - Universidade Federal de Pernambuco

Av. Moraes Rego, 1235 Cidade Universitária 50.670-901 Recife PE Brasil

Phone/Fax: 00-55-81-21268522

E-mail: [email protected] Web site: www.ufpe.br/nem

Running head: Aroma production by Dekkera bruxellensis for cachaça

This article has been accepted for publication and undergone full peer review but has not been through the copyediting typesetting pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/yea.3051

mailto:[email protected]

http://www.ufpe.br/nem


Abstract

The distilled spirit made from sugarcane juice also known as cachaça is a traditional Brazilian

beverage that in recent years has increased its market share among international distilled

beverages. Several volatile compounds produced by yeast cells during the fermentation

process are responsible for the unique taste and aroma of this drink. The yeast Dekkera

bruxellensis has acquired increasing importance in the fermented beverage production, as the

different metabolites produced by this yeast may be either beneficial or harmful to the end-

product. Since D. bruxellensis is often found in the fermentation processes carried out in

ethanol fuel distillation in Brazil, we employed this yeast to analyse the physiological profile

and production of aromatic compounds and examine whether it is feasible to regard it as a

cachaça-producing microorganism. The assays were performed on a small scale, and

simulated the conditions for the production of handmade cachaça. The results showed that the

presence of aromatic and branched-chain amino acids in the medium has a strong influence

on the metabolism and production of flavours by D. bruxellensis. The assimilation of these

alternative nitrogen sources led to different fermentation yields and the production of

flavouring compounds. The influence of the nitrogen source on the metabolism of fusel

alcohols and esters in D. bruxellensis highlights the need for further studies of the nitrogen

requirements to obtain the desired level of sensory compounds in the fermentation. Our

results suggest D. bruxellensis has the potential to play a role in the production of cachaça.

Keywords: cachaça, alcoholic fermentation, fusel alcohols, volatile esters, Ehrlich pathway,

nitrogen source.


1. Introduction

Cachaça is a handmade alcoholic beverage in Brazil that is produced by distilling

fermented sugarcane juice. The fermentation occurs in the presence of high yeast cell counts

(109 cells/mL, approx. 10-12% w/v), high CO2 pressure, temperatures ranging from 30 to 33

ºC and in the absence of agitation (Vila Nova et al., 2009; Vidal et al., 2013). Ethanol and

CO2 are the main compounds produced during this process, together with other minor

metabolites that play an important role in the aroma and taste (Nascimento et al., 2008). The

fusel alcohols and volatile esters are the main compounds responsible for providing the

desired aroma in alcoholic beverages (Vidal et al., 2013; Verstrepen et al., 2013). Fusel

alcohols can be produced by carbohydrate dissimulation and also by the catabolism of

aromatic or branched-chain amino acids (BCAA) in the Ehrlich pathway (Hazelwood et al.,

2008). Some of the most important fusel alcohols that have organoleptic properties are (i)

isoamyl alcohol (3-methyl-1-butanol), (ii) active amyl alcohol (2-methyl-1-butanol), (iii)

isobutanol (2-methyl-1-propanol), (iv) tryptophol (indole-3-ethanol), (v) tyrosol, and (vi)

phenylethanol (2-phenyl-ethanol), derived from the catabolism of leucine, isoleucine, valine,

tryptophan, tyrosine and phenylalanine, respectively (Hazelwood et al., 2008). Additionally,

acyl esters are produced by transesterification reactions between an alcohol and a carboxylic

acid (Verstrepen et al., 2013). The principal ester found in cachaça is ethyl acetate

(Nascimento et al., 2008).

The number of these organoleptic compounds in cachaça depends on several factors,

including the fermentation conditions, the nature of the nitrogen source and the yeast strain

employed in the process (Vila Nova et al., 2009; Vidal et al., 2013; de Souza et al., 2012;

Duarte et al., 2013). Several yeasts and bacteria are present in the microbial population of

handmade cachaça and these species are likely to act together to produce metabolites which


might be either advantageous or detrimental to the process and determine the sensory quality

of the end-product (Vila Nova et al., 2009; Duarte et al 2011; Duarte et al., 2013; Gomes et

al., 2010). Several yeasts have been employed in the production of fermented alcoholic

beverages, and this has resulted in the formation of several different compounds (Vila Nova

et al., 2009; Duarte et al 2013). The use of selected yeast starters is a practice that is common

in the wine and beer industries, but not yet applied to production of cachaça. Most distilleries

that produce cachaça rely on spontaneous fermentation, which is a process that depends on

the microbiota found in the environment (sugar cane). Previous studies have shown there are

several species of wild yeast in the spontaneous fermentation, although the population of

Saccharomyces cerevisiae is predominant (Morais et al, 1997; Schwan et al, 2001; Vila Nova

et al., 2009). Other species that can be found are Pichia, Candida, Zygosaccharomyces,

Lachancea, Rhodotorula and Schizosaccharomyces (Morais et al, 1997; Schwan et al. 2001;

Vila Nova et al., 2009). So far, there have been no reports of the presence of Dekkera

bruxellensis in this process, despite its adaptation to fuel ethanol fermentation (De Souza

Liberal et al. 2007). The fact that many other yeast species have the potential to produce a

particular range of flavours might allow a different consortium of microorganisms to be used

for the production of particular beverages.

The yeast Dekkera bruxellensis (teleomorph of Brettanomyces bruxellensis) is known

worldwide as major contaminant yeast in wineries (Zuehlke et al., 2013). Its negative effect

in winemaking is caused by the production of volatile phenols regarded as off-flavours, such

as 4-ethylphenol and 4-ethylguaiacol. These are responsible for a phenomenon known as

“Brett”, which is easily recognizable from spoiled wines and described as “Band-aid”,

“barnyard-like”, “horsey”, “sweaty”, “wet dog”, among other expressions, and is a cause of a

financial loss to the industry (Snowdon et al., 2006; Sangorrín et al., 2013). This species is

able to consume a wide range of carbon and nitrogen sources. Glucose and sucrose lead to the


highest cell growth rates (Leite et al., 2013), the latter being the main component of sugar in

sugarcane juice which acts as a substrate for the production of cachaça. With regard to the

nitrogen sources, this yeast is able to make use of ammonium, nitrate, urea and amino acids

(Blomqvist et al., 2012; de Barros Pita et al., 2013a; de Barros Pita et al., 2013b). In addition,

in D. Bruxellensis, nitrate assimilation has been cited as an adaptation strategy in bioethanol

production in Brazilian distilleries (de Barros Pita et al., 2011). Dekkera bruxellensis has also

been successfully used in the production of alcoholic beverages, since this yeast has long

been used in the production of Belgian Lambic beer (Martens et al., 1997; Daenen et al.,

2008). Recently, we reported the presence of two paralogs of the ARO10 gene in the genome

of this yeast (De Souza Liberal et al., 2012), which suggests that D. bruxellensis has the

capacity to catabolize branched-chain and aromatic amino acids for the production of sensory

compounds through the Ehrlich pathway (Hazelwood et al., 2008).

Since D. bruxellensis often forms a part of the microbiota in the distilleries that

ferment sugarcane juice in Brazil (de Souza Liberal et al., 2007; Basílio et al., 2008) and

participates in the production of alcoholic beverages, we investigated whether this yeast

could be used in the production of handmade cachaça by exploring its capacity to produce

fusel alcohols derived from aromatic and branched-chain amino acids. For this reason, we

evaluated the influence of different nitrogen sources on the production of these alcohols and

discussed the prospect of using D. bruxellensis in the production of the most important

Brazilian alcoholic beverage.

2. Material and Methods

2.1. Yeast strains and growth conditions

The industrial strain Dekkera bruxellensis GDB 248 (de Barros Pita et al., 2011; Leite

et al., 2013) was used in this study. Cells were pre-grown in 500 mL flasks containing 150


mL of YPS medium (Yeast Extract, 10 g/L; peptone, 20 g/L; sucrose, 20 g/L). Cells were

grown at 30°C and 160 rpm in a rotator shaker for 72 h to produce yeast biomass for

fermentation assays.

Fermentation experiments were performed in laboratory media with compounds

containing different nitrogen sources. YPS medium used for fermentation differs from the

growth medium which has a sucrose content of 120 g/L. The mineral medium (MMS)

contained a complete Yeast Nitrogen Base (YNB) at 6.7 g/L and sucrose at 120 g/L (12%

w/v). The MMS+AA and MMS+ARO media contained YNB (w/o amino acids and

ammonium sulphate) at 1.7 g/L and sucrose at 120 g/L supplemented with a mixture of

branched-chain (leucine, isoleucine and valine) or aromatic amino acids (phenylalanine,

tyrosine and tryptophan), respectively, at 0.40 g of nitrogen per litre of medium (for each N

source). The absence of the sulphate ion was offset by the addition of an equimolar amount of

potassium sulphate (Table 1). Sugarcane juice was used as an industrial substrate for cachaça

production and obtained from local producers. In brief, the sugarcane was crushed and the

juice (SCJ) was collected on ice, sent to the laboratory, clarified by serial filtrations, sterilized

at 121ºC for 15 minutes and stored at 4ºC until use (Vidal et al., 2013). The sucrose content

was determined by HPLC and before it was used, SCJ was diluted with sterile distilled water

to sucrose at 120 g/L. All of the fermentation media were supplemented with nalidixic acid

(50 mg/mL) and ampicillin (50 µg/mL) to prevent bacterial contamination.

2.2 Fermentation assays and cell viability

Cells were pre-grown on YPS, harvested by centrifugation (5 min, 5.000 g, at room

temperature) in 50 mL conical tubes and pooled until the equivalent of 6 g of cells (wet

weight) were obtained. Following this, the tubes were filled with 50 mL of each of the

fermentation media. The cell suspensions were transferred to 125 mL flasks, which resulted


in a final biomass concentration of around 100 g/L (1-2 x 109 cells/mL), similar to that found

in industrial processes. Fermentation experiments were carried out in biological replicates at

32ºC under slight agitation (50 r.p.m. orbital agitation) to prevent cell sedimentation. The cell

density and viability were determined through an assessment of the cells stained with

methylene blue by means of an optical microscope and using a Neubauer chamber. Samples

of 1.5 mL were withdrawn in technical replicates at the beginning of the fermentation, every

hour for 12 hours and at the end of the fermentation (24 h). The supernatant was collected

after centrifugation (11.000 rpm for 1 min at 4 ºC) and stored at -20 ºC until metabolic

analysis (HPLC and GC).

2.3 Metabolite analysis by HPLC and GC

Four samples (two technical replicates from two biological replicates) for each

fermentation time were collected for each media. Glucose, fructose, sucrose, acetate, ethanol,

and glycerol were measured by HPLC on the Agilent 1100 Series platform (Agilent, USA)

equipped with an ionic exchange column, HPX-87H (Biorad, USA) at 35ºC and using 5 mM

H2SO4 as mobile phase with a flow of 600 µL/min. Ethyl acetate, 1-propanol, isobutanol,

butanol, amyl alcohol and isoamyl alcohol were evaluated by gas chromatography (GC)

using a Restek column (60 mm x 0.32 mm x 1.0 mm) and hydrogen as mobile phase. The

injection temperature was 250ºC, the initial and final temperatures of the oven were 40ºC (6.5

min) and 103ºC, respectively. The transport gas flow was 7 mL/min and the temperature of

the detector was adjusted to 90ºC. The volume of sample injected was 10 µL. All the

compounds measured were standardized with 2-pentanol (internal control), defined in terms

of their retention times and quantified by means of a standard curve for each compound

(Sigma-Aldrich Co.). The standard error was determined as below 10% for all the

measurements. Active amyl alcohol and isoamyl alcohol were indistinguishable in the GC


methods employed here and were referred to as AI portion. 2-phenylethanol was evaluated by

means of gas chromatography (GC) using DB-WAX column (30 m x 0.325 mm) with helium

as a transport gas and an ionization detector. The injection temperature was 250ºC, the initial

and final temperatures in the oven were 40ºC (7 min) and 200ºC, respectively. The transport

gas flow was 30 mL/min and the volume of sample injected was 4 µL.

3. Results and Discussion

3.1. Fermentation parameters

The potential application of D. bruxellensis for cachaça production was assessed by

examining its metabolic and organoleptic profile in fermentation assays with different

nitrogen sources, in conditions similar to those of industry (with high sugar concentrations

and yeast biomass). The yeast population almost doubled on MMS, YPS and SCJ medium

(Fig. 1) until the end of the fermentation, which correlates with the amount of glycerol

production observed and the high level of cell viability, which was over 90% (Table 1). The

tendency for D. bruxellensis cells to grow under oxygen-limited conditions to assure full

fermentation has been reported recently (Pereira et al., 2012; Blomqvist et al., 2012; Leite et

al., 2013). It should be noted that ammonium was the nitrogen source in the MMS mineral

medium, while a mixture of ammonium and nitrate was found in SCJ medium (Table 1).

Glycerol is commonly combined with biomass formation through the oxidation of

intermediary metabolites to produce amino acids. Moreover, yeasts produce glycerol as a

fermentation by-product under oxygen limitation so that they can re-oxidize the NADH that

has been produced (Van Dijken and Scheffers, 1986). A typical trait described in yeasts of

the Dekkera/Brettanomyces genera is their inability to produce glycerol under anaerobiosis,

which leads to redox imbalance that slows down glycolysis and fermentation - the so-called


Custers effect (Wijsman et al., 1984). However, several studies have reported glycerol

production by D. bruxellensis under low-oxygen conditions, even though at lower levels than

S. cerevisiae (Aguilar Uscanga et al., 2003; de Souza Liberal et al., 2007; Pereira et al., 2012;

de Barros Pita et al., 2013a). In the present study, the glycerol yield in SCJ was lower than

that in YPS and MMS laboratory media (Table 1). This could be caused by the presence of

nitrate in the sugarcane, which is liable to divert NADH produced in the glycolytic pathway

to its reductive assimilation to ammonium. It could abolish the Custers effect (de Barros Pita

et al., 2011; Galafassi et al., 2013a). Thus, traces of nitrate may induce a

respiratory/oxidative state in the D. bruxellensis cells even under oxygen-limited conditions

in the presence of high sugar content. On the other hand, no growth was detected in

MMS+AA medium (Fig. 1) and a reduction in cell viability was also observed (Table 1).

Despite this, a small amount of glycerol was produced, which is suggestive of metabolic

disturbance caused by the excess of branched chain amino acids (BCAA) and its catabolic

products (Table 1). Glycerol production was recently reported for D. bruxellensis under

osmotic stress (Galafassi et al., 2013b).

A complete sucrose consumption was observed in SCJ medium at the end of 24 h

fermentation, followed by partial sugar assimilation in MMS+ARO, YPS, MMS and

MMS+AA (Table 1 and Fig. 2A). The highest sucrose assimilation rate in SCJ (Table 1)

could again be explained by the presence of nitrate in the sugarcane juice. When in

combination with ammonium, it is found that nitrate stimulates cell growth, sucrose

consumption and ethanol production in D. bruxellensis cells (de Barros Pita et al., 2011;

Galafassi et al., 2013a). Thus, the presence of nitrate could induce oxidative metabolism in

this yeast, which in part explains the high rate of acetate production (Table 1 and Fig. 2C). In

fact, acetate production is closely linked to respiratory metabolism in D. bruxellensis and is

often found to be the main fermentation product in aerobiosis (Leite et al., 2013; Galafassi et


al., 2013a). This means that its production in YPS and MMS can be attributed to the presence

of an alternative electron acceptor (Fig. 2C). The fact that the presence of this kind of

compound can stimulate acetate production might explain the acidic taste of Lambic beer,

and might also be a contributory factor for the sensory perception of acidity in the cachaça

that was eventually produced by D. bruxellensis.

High residual sugar was detected in MMS+AA, and 53% of initial sucrose

concentration remained in the medium at the end of the fermentation assay (Table 1; Fig.

2A). As a result, the production of ethanol was lower than that of the other nitrogen sources

and severely affected the ethanol yield (Table 1; Fig. 2B). Thus, the excess of BCAA in the

medium impaired the fermentation performance of the D. bruxellensis cells. BCAA are

usually regarded as secondary nitrogen sources and are only assimilated in the absence of a

preferred source, such as ammonium or glutamine. In addition, they are well known for their

low assimilation rate and for causing slower cell growth (Magasanik and Kaiser, 2002; Boer

et al., 2007; De Barros Pita et al., 2013a). In this study, we also observed a loss of cell

viability in the presence of these amino acids. When this is taken into account, together with

the slow assimilation rate of sucrose, it may explain the lower ethanol productivity achieved

by D. bruxellensis cells (Table 1). Moreover, the formation of toxic -aldehydes derived

from BCAA catabolism, might create a large demand for NADH in reductive reactions with

regard to fusel alcohol production (see below).

In the MMS+ARO medium, cell biomass was increased by 40% at the end of the

fermentation assay (Fig. 1) along with an increase in the consumption of sucrose (Fig. 2A)

and the highest final ethanol concentration compared with other media (Table 1). The fact

that the biomass formation was lower than the MMS, YPS and SCJ media, can be explained

by the presence of ethanol and 2-phenylethanol in the medium, which were reported as

interfering with the yeast physiology (Serp et al., 2003).


3.2. Production of sensory compounds

The metabolic profile of D. bruxellensis was investigated under different nitrogen

sources (Table 2 and Fig. 3) and the most important flavouring compounds in distilled

beverages were investigated. With regard to the industrial substrate (SCJ), the AI portion was

12 times lower than those found for S. cerevisiae in the same fermentation conditions (Vidal

et al., 2013). However, it was very close to the production of AI portion reported by Dato et

al. (2005) from a D. bruxellensis strain isolated from the fermentation process in Southern

Brazil. In that study, the authors defined the concentrations as being the amount of

metabolites per 100 mL of distilled product. Thus, we were able to correct those results by

taking into account the concentration factor of distillation. By adopting the same strategy, we

calculated that D. bruxellensis GDB 248 produced AI portion in the same range as Pichia

caribbica in SCJ medium, a species reported as having the potential to co-ferment yeast for

cachaça production (Vila Nova et al., 2009; Duarte et al., 2013), and other non-S. cerevisiae

yeasts isolated from cachaça fermentation (Oliveira et al., 2005; Vila Nova et al., 2009;

Duarte et al., 2013). The production of isobutanol by D. bruxellensis GDB 248 was 10 times

higher in SCJ medium than with non-S. cerevisiae strains isolated from fermentation

processes (Oliveira et al., 2005; Vila Nova et al., 2009; Duarte et al., 2013). As expected, the

production of fusel alcohols derived from the BCAA was higher in MMS+AA medium than

in other media (Table 2). In this condition, the AI portion increased more than 10 times that

of YPS, MMS, MMS+ARO or SCJ (Table 2 and Fig. 3A), while isobutanol in MMS+AA

only increased twice as much as MMS, YPS and SCJ media (Fig. 3B). No isobutanol was

detected in the MMS+ARO medium (Table 2 and Fig. 3B).

In spite of having the same initial concentration of nitrogen for the three amino acids

in MMS+AA medium (0.40 g of N/L), the production of AI portion derived from leucine and


isoleucine, was almost 7 times higher than the production of isobutanol derived from valine

(Table 2 and Fig. 3A-B). Moreover, the production of AI portion in the medium started at the

beginning of the fermentation, while the isobutanol was only detected after 8 hours of

fermentation (Fig. 3A-B). Overall, our data suggest that D. bruxellensis is able to use

branched-chain amino acids as nitrogen sources although at different assimilation rates. It is

hoped that our ongoing physiological experiments will enable us to quantify these

preferences. This difference in the assimilation can be explained by the different affinities

displayed by the α-ketoacid decarboxylases. In S. cerevisiae, the three main decarboxylases

isoenzymes of S. cerevisiae encoded by the genes PDC1, PDC5 and ARO10, show different

affinities for the substrates derived from BCAA catabolism, (α-ketoisocaproate derived from

leucine, α-ketoisovalerate derived from valine and α-keto-β-methylvalerate derived from

isoleucine) (Vuralham et al., 2005; Romagnoli et al., 2012). Hence, our data suggest that D.

bruxellensis has decarboxylases with similar affinities to their homologues in S. cerevisiae

which might explain the lower production of isobutanol in MMS+AA. In addition, the

enzyme phenylpyruvate decarboxylase encoded by the ARO10 gene, also has a high affinity

for these α-ketoacid substrates (Vuralham et al., 2005; Romagnoli et al., 2012). It is worth

noting that the D. bruxellensis genome carries two paralogs of the ARO10 gene, a particular

trait of this yeast among the ascomycetes (de Souza Liberal et al., 2012). A high production

of 2-phenylethanol (in the range of 555 mg/L) was observed in MMS+ARO medium, while

only trace amounts were detected in the other media tested. Previous works have shown the

biotechnological potential of Kluyveromyces marxianus and S. cerevisiae yeasts for the

production of 2-phenylethanol from L-phenylalanine (Fabre et al., 1998; Hazelwood et al.,

2008). Our results suggest that D. bruxellensis may be included in this group of 2-

phenylethanol producers. This compound has a characteristic flavour and fragrance that has

many biotechnological applications (Hazelwood et al., 2008). Thus, the genetic and


physiological capacity of D. bruxellensis for producing 2-phenylethanol could be further

explored.

The production of 1-propanol was in inverse proportion to the nitrogen concentration

in the medium: higher in SCJ medium (171 mg N/L) and lower in YPS medium (5500 mg

N/L), and intermediary in MMS medium (1059 mg N/L) (Table 2). Moreover, it started

earlier in the SCJ medium (Fig. 3B). This profile of 1-propanol production was previously

observed in S. cerevisiae strains in the winemaking industry (Carrau, 2008). However, no

production of 1-propanol was observed in the MMS+AA and MMS+ARO media (Table 2).

1-propanol is one of the main components of fusel alcohol, but unlike the other metabolites

that are derived from amino acid catabolism, it is formed by the condensation of pyruvic acid

and acetyl-CoA (Hazelwood et al., 2008). This draws attention to the fact that 1-propanol

was not detected in MMS+AA medium (Fig. 3C), which may be because the available supply

of acetyl-CoA is smaller in this medium, which is corroborated by the absence of acetate

(Table 1).

With regard to ethyl acetate, we observed its production was higher in SCJ and

MMS+ARO and lower in MMS+AA (Table 2 and Fig. 3D). This can again be explained by

the availability of acetate, and hence acetyl-CoA, which is similar to the case of 1-propanol

production discussed above. In S. cerevisiae, ethyl acetate is the main acetate ester produced

when fermenting SCJ (Vidal et al., 2013). However, D. bruxellensis produced only half of

the amount observed in S. cerevisiae. Ethyl acetate is formed by the condensation of one

molecule of ethanol and one acetyl-CoA. Since acetate is not detected in this medium, less

acetyl-CoA is likely to be available for esterification and ethyl acetate formation. In addition,

the production of this ester by S. cerevisiae in brewing is the result of a balance between acyl

transferases and esterase enzymes encoded by EEB1 and IAH1 genes, respectively (Saerens

et al., 2006). When fermenting SCJ, S. cerevisiae showed an up-regulation of EEB1 and


down-regulation of IAH1 gene (unlike when this substrate was supplemented with

ammonium sulphate), and this led to a higher production of the ester (Vidal et al., 2013). The

expression of their homologues in D. bruxellensis must be analysed in future work to

determine what biological roles they play in the metabolism of ester formation in D.

bruxellensis.

3.3 Concentration of the metabolites in cachaça

Brazilian legislation lays down the quality standards that must be complied with in the

production of cachaça (Brazil, 2005). Table 3 shows the maximum values permitted by

legislation for the compounds analyzed in this work. The purpose of the established

legislative standards is to moderate the influence of each of these compounds as a means of

protecting public health and the quality of the drink (Miranda et al., 2007). The composition

of the yeast population is an important parameter for the quality of the cachaça produced

(Vila Nova et al., 2009). During the spontaneous fermentation of the cachaça the continuous

introduction of new strains from the sugarcane juice and the non-sterile process occurs, and

as a result, there is a wide variation in their chemical composition (Morais et al., 1997;

Miranda et al., 2007; Fernandes et al., 2004).

Miranda et al (2007) analyzed the chemical compounds in 94 samples of commercial

cachaças that complied with the quality standards set out in current legislation and observed

significant variations in their composition. The concentration of higher alcohols in mg/100

mL ethanol varied between 443.35 and 152.46, while the concentration of ethyl acetate

ranged from 418.85 to 0.97 (Miranda et al., 2007). Thus, some of these samples exceeded the

maximum limit permitted by law (Table 3). Fernandes et al (2004) also found considerable

differences in the chemical composition of different samples of cachaça produced in Brazil,

after analyzing 16 Brazilian brands. The values of the chemical components of samples

evaluated in this study, when the distillation factor was applied, do not exceed the limits


stipulated by law (Table 3). The production of ethyl acetate was higher in the SCJ medium

and MMS+AA medium, while the production of higher alcohols was only comparable with

the results of Miranda et al (2007) in the middle MMS+AA (Table 3). This provides evidence

that when sugarcane juice is supplemented with branched amino acids, it can exert a strong

influence on the sensory quality of the product.

4. Conclusion

In this study, we evaluated the physiological profile of D. bruxellensis and the

production of volatile compounds which have organoleptic characteristics in response to

different nitrogen sources. Our results showed that in conditions similar to those found in the

production of cachaça, the supplementation of media with aromatic and branched-chain

amino acids exerts a strong influence on the production of aromas by D. bruxellensis. This

yeast can use these amino acids as nitrogen sources, although they have different

fermentative capacities from the preferred nitrogen sources, such as ammonium. This could

be either due to a slow rate of sucrose assimilation or the inhibitory effect of fusel alcohols,

or their intermediate toxic compounds. Since D. bruxellensis is capable of producing sensory

compounds and is highly adaptable to industry, this study might be of value to the industrial

application of this yeast in the production of cachaça.


Acknowledgements

This work was supported by grants from the CNPq-FACEPE program (PRONEM Project

No. APQ-1452-2.01/10) and was carried out within the framework of COST Action FA0907

BIOFLAVOUR (www.bioflavour.insa-toulouse.fr) under the EU Seventh Framework

Programme for Research and Technological Development (FP7). DCP would like to express

its thanks to the FACEPE agency for its doctoral scholarship support.

Conflict of interest: The authors declare no conflict of interest.

Compliance with ethical requirements: this article does not contain any studies with human

or animal subjects.

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Figure 1. Biomass variation of Dekkera bruxellensis GDB 248 strain during fermentation of

sugarcane juice (diamonds), YPS (triangle), MMS (square), MMS+AA (open circle) and

MMS+ARO (closed circle). The results represent the mean value of two biological replicates

and two technical replicates for each point.


Figure 2. Physiological profile of Dekkera bruxellensis GDB 248 strain in different media

for consumption of sucrose (panel A) and production of ethanol (panel B), acetate (panel C)

and glycerol (panel D) during fermentation of sugarcane juice (diamonds), YPS (triangle),

MMS (square), MMS+AA (open circle) and MMS+ARO (closed circle).


Figure 3. Kinetics of the production of AI portion (straight lines) and 2-phenylethanol

(dotted line) (panel A), isobutanol (panel B), 1-propanol (panel C) and ethyl acetate (panel D)

by Dekkera bruxellensis GDB 248 strain during fermentation of sugarcane juice (diamonds),

YPS (triangle), MMS (square), MMS+AA (open circle) and MMS+ARO (closed circle).


Tab

le 1

. P

hysi

olog

ical

par

amet

ers

of t

he f

erm

enta

tion

of

suga

rcan

e ju

ice

(SC

J),

com

plex

med

ium

(Y

PS

), m

iner

al m

ediu

m (

MM

S)

and

min

eral

med

ium

sup

plem

ente

d w

ith

bran

ched

-cha

in (

MM

S+

AA

) or

aro

mat

ic (

MM

S+

AR

O)

amin

o ac

ids

by D

ekke

ra b

ruxe

llen

sis

GD

B 2

48 s

trai

n.

Nit

roge

n

Suc

rose

Eth

anol

Gly

cero

l

Ace

tate

Med

ia

Sou

rce

g/L

FA

N

(mgN

/L)

In

itia

l

(g/L

)

Fin

al

(g/L

)

Qs

(g/L

.h)

F

inal

(g/L

)

Yie

ld

(g/g

)

Qp

(g/L

. h)

F

inal

(g/L

)

Yie

ld

(g/g

)

Qp

(g/L

.h)

F

inal

(g/L

)

Yie

ld

(g/g

)

Qp

(g/L

. h)

SC

J A

mm

oniu

m

0.57

17

1.09

118.

54

0.00

-4

.94

34

.02

0.29

1.

42

1.

94

0.02

0.

08

3.

06

0.03

0.

13

Nit

rate

0.

24

YP

S

Pep

tone

20

.00

5500

.00

12

1.16

16

.57

-4.3

6

39.2

7 0.

38

1.64

3.01

0.

03

0.13

1.23

0.

01

0.05

MM

S

Am

mon

ium

5.

00

1059

.00

11

2.68

42

.46

-2.9

3

30.1

6 0.

43

1.26

3.06

0.

04

0.13

1.14

0.

02

0.05

MM

S+

AA

Leu

cine

3.

84

1229

.00

108.

04

57.6

6 -2

.10

7.59

0.

15

0.32

0.60

0.

01

0.03

0.00

0.

00

0.00

V

alin

e 3.

84

Isol

euci

ne

3.84

MM

S+

AR

O

Phe

nyla

lani

ne

Tyr

osin

e

Try

ptop

han

0.41

0.41

0.41

1230

.00

11

6.25

0.

90

-4.8

4

46.3

0.

40

1.93

1.48

0.

01

0.06

2.82

0.

03

0.12


Table 2. Final concentration and maximum productivity (Pmax) of volatile compounds

produced by Dekkera bruxellensis GDB 248 during fermentation of sugarcane juice (SCJ)

and laboratory media YPS, MMS, MMS+AA and MMS+ARO media.

Ethyl acetate Propanol Isobutanol AI Portion

Final Pmax

Final Pmax

Final Pmax Final Pmax

Media (mg/L) (mg/L.

h) (mg/L) (mg/L.h)

(mg/L) (mg/L.h)

(mg/L) (mg/L.h)

SCJ 111.83 4.66

11.23 0.47

19.15 0.8

18.21 0.76

YPS 55.89 2.33

4.84 0.2

20.33 0.85

17.32 0.72

MMS 77.69 3.24

8.48 0.35

17.87 0.74

21.11 0.88

MMS+AA 18.02 0.75

0 0

35.67 1.49

248.53 10.36

MMS+ARO 127.47 5.31 0 0 0 0 25.81 1.07


Table 3. Final concentration of volatile compounds produced by Dekkera bruxellensis GDB

248 in the fermentation must and after distillation and maximum concentration permitted by

Brazilian Legislation.

Ethyl acetate IA portion+propanol+isobutanol

Media Distillate

(mg/100 mL)

Maximum allowed

(mg/100 mL)a

Distillate

(mg/100 mL)

Maximum allowed

(mg/100 mL)a

SCJ 67.1

200

29.154

360

YPS 33.5 25.494

MMS 46.6 28.476

MMS+AA 10.8 170.52

MMS+ARO 76.5 15.486

aSource: Brazil, 2005 (standard ruling , Ministry of Agriculture)

Production of sensory compounds by means of the yeast Dekkera bruxellensis ...

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