Food Chemistry 138 (2013) 701708

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Inuence of nitrogen supply on the production of higher alcohols/esters and expression of avour-related genes in cachaa fermentation
Esteban Espinosa Vidal a,e,f,, Gustavo M. de Billerbeck e, Diogo Ardaillon Simes a,b, Alexandre Schuler c, Jean Marie Franois e, Marcos Antonio de Morais Jr. a,b,d
a

Interdepartmental Research Group in Metabolic Engineering, Federal University of Pernambuco, Av. Moraes Rego 1235, 50670-901 Recife, Pernambuco, Brazil Department of Biochemistry, Federal University of Pernambuco, Av. Moraes Rego 1235, 50670-901 Recife, Pernambuco, Brazil Department of Chemical Engineering, Federal University of Pernambuco, Av. Moraes Rego 1235, 50670-901 Recife, Pernambuco, Brazil d Department of Genetics, Federal University of Pernambuco, Av. Moraes Rego 1235, 50670-901 Recife, Pernambuco, Brazil e Universit de Toulouse, INSA, UPS, INP, INRA-UMR792 & CNRS-UMR 5504, Ingnierie des Systmes Biologiques et des Procds, 135 Av. de Rangueil, F-31077 Toulouse, France f Center for Strategic Technologies of Northeast CETENE, Av. Prof. Luiz Freire 01, 50740-540 Pernambuco, Brazil
b c

a r t i c l e

i n f o

a b s t r a c t
This study provides the rst attempt to analyse the inuence of ammonium supplements on sugar-cane juice fermentation and the avour prole in a cachaa industrial process. The objective was to nd a relationship between higher alcohol/ester content and the transcription levels of the main genes involved in production of these compounds under cachaa fermentation. Sugar-cane juice with a low amount of assimilable nitrogen (81 mg N/L), was further supplemented with mid-range or high concentrations of ammonium sulfate. Overall, higher alcohol production was reduced by ammonium supplementation, and this can be correlated with a general downregulation of genes encoding decarboxylases and dehydrogenases of the Ehrlich pathway. The production of acetate esters was enhanced by mid-range ammonium supplementation and the production of acyl esters by high ammonium supplementation. The acyl esters could be correlated with expression of alcohol acyl-transferase EEB1 and the acyl esterase IAH1. 2012 Elsevier Ltd. All rights reserved.

Article history: Received 25 July 2012 Received in revised form 16 October 2012 Accepted 30 October 2012 Available online 10 November 2012 Keywords: Cachaa Ehrlich pathway RT-qPCR Sensorial compounds

1. Introduction Cachaa is the main Brazilian white spirit beverage that is obtained from the distillation of sugar cane juice fermented by the yeast Saccharomyces cerevisiae (de Arajo Vicente, Fietto, de Miranda Castro, Coutrim, & Brandao, 2006). Several unique fermentative parameters characterise the production of cachaa: the concentrations of sugars, principally in the form of sucrose in the sugar cane juice is in the range of 120240 g/L, the content of free amino nitrogen level is around 100 mg/L and directly depends on the type of cane cultivar and agronomical conditions. The fermentation occurs at a very high biomass concentration (in the order of 109 cells/ mL, approx. 1012% cell w/v), under high carbon dioxide pressure to ensure conditions close to anaerobiosis, at high temperature (between 30 and 33 C) with no agitation of the medium. All of these features make the manufacture of cachaa a very specic
Abbreviations: SCJ, sugar cane juice; MAS, midst ammonium supplementation; HAS, high ammonium supplementation. Corresponding author at: Interdepartmental Research Group in Metabolic Engineering, Federal University of Pernambuco, Av. Moraes Rego 1235, 50670-901 Recife, Pernambuco, Brazil. Fax: +55 81 2126 8522. E-mail addresses: esteban.espinosa.vidal@gmail.com, esteban.vidal@cetene. gov.br (E.E. Vidal). 0308-8146/$ - see front matter 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.10.147

industrial practice that may represent some scientic interest with respect to the fermentation process. During the fermentation, the yeast cells produce a broad spectrum of volatile molecules, such higher alcohols, aldehydes, organic acids, esters, organic suldes, and carbonyl compounds (Hazelwood, Daran, van Maris, Pronk, & Dickinson, 2008) that confer sensory characteristics on the beverages. Higher alcohols and esters are of major industrial interest because of their high concentrations (with respect to other secondary metabolites) and aroma contribution (Francis & Newton, 2008; Procopio, Qian, & Becker, 2011). Higher alcohols are produced from aromatic and branched-chain amino acids (BCAA), through the catabolic Ehrlich pathway (Ehrlich, 1907), or from pyruvate, through 2-oxoacid intermediates involved in the biosynthesis of the BCAA (Hazelwood et al., 2008). In the catabolic pathway, the amino acids are rst transaminated to 2-oxoacids, which in turn are decarboxylated to aldehydes and nally reduced to the corresponding alcohols. Typical concentrations of higher alcohols in beer are in the range of 100200 mg/L (Quain & Dufeld, 1985) and in wine in the range of 244 mg/L (Valero, Moyano, Millan, Medina, & Ortega, 2002). The other important avours for dening the sensorial quality of spirit beverages are acetate and ethyl esters, since they are

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characterised by fruity and oral aromas (Verstrepen et al., 2003). Acetate esters are formed by the condensation of acetyl-CoA with an alcohol, such as isoamyl acetate and isobutyl acetate, whereas acyl esters, such as ethyl hexanoate (ethyl caproate) and ethyl octanoate (ethyl caprylate) (Dufour, Malcorps, & Silcock, 2003; Meilgaard, 2001), are produced by the condensation of CoAactivated medium-chain-fatty acids with ethanol. The concentrations of esters vary widely in alcoholic beverages (Boulton & Quain, 2001; Saerens et al., 2008; Valero et al., 2002). With respect to cachaa production, it is widely accepted by both artisanal and industrial producers that adding ammonium sulfate to the sugar cane broth can help to maintain high fermentation rates and elevated yeast populations during the fermentation process (Angelis & Mutton, 1992). It has been widely reported that the nature of the nitrogen source and its availability strongly determine the nal content of aromas and avours in the fermented must (Bell & Henschke, 2008; Carrau et al., 2008; Hazelwood et al., 2008; Lambrechts & Pretorius, 2000; OConnor-Cox and Ingledew, 1989; Torrea-Goni & Ancin-Azpilicueta, 2002; Wang, Bohlscheid, & Edwards, 2003). For example, excessive wort amino nitrogen tends to elevated higher alcohol concentrations by the stimulatory effect in cellular growth. On the contrary, very low amino acid nitrogen also yields an excessive higher alcohol concentration (Szlavko, 1974). In this instance, the limited nitrogen content will promote enhanced endogenous synthesis of amino acids, stimulating the anabolic higher alcohol synthetic route (Boulton & Quain, 2001). However, in wine, the production of higher alcohols tends to decrease with an increase in nitrogen supplementation (Carrau et al., 2008). It has been reported that the C/N ratio (Palmer & Rennie, 1974; Younis & Stewart, 1999), pitching rates (Maule, 1967), and lipid synthesis activity (Boulton & Quain, 2001) inuence the ester concentration in the medium. One accepted factor that regulates ester formation is the availability of acetyl-CoA. Any change in the size of the acetyl-CoA pool should have an effect on the ester concentration. Saerens et al. (2008) showed that increased carbon content or a higher level of nitrogen cause a stronger increase in acetate ester production than in ethyl ester production. Although there are several studies relating the microbial diversity and the aroma prole of cachaa (De Arajo Vicente et al., 2006; Gomes, Silva, Marini, Oliveira, & Rosa, 2007; Oliveira, Cardello, Jeronimo, Souza, & Serra, 2005; Oliveira et al., 2008; Vilanova, Schuler, Brasileiro, & Morais, 2009), there are no studies that address how ammonium supplementation, a common practice in cachaa industrial production, affects the production of higher alcohol and esters. The purpose of this work was to carry out an investigation of the changes in avour proles that are caused by nitrogen supplementation in cachaa manufacture, with regard to the expression of principal genes encoding enzymes involved in the biosynthesis of higher alcohols and esters.

transported to the laboratory where it was immediately claried by rapid boiling for ve minutes and then stored for 12 h at 4 C for decantation. Afterwards, the supernatant was ltered by lter paper, sterilised at 121 C for 15 min, and stored at 4 C. Before use, the sugar cane juice was adjusted to 12 Brix (ca. 120 g sucrose/L) with sterile water, as performed by cachaa producers. Sugar cane fermentation must (SCJ) was supplemented with nalidixic acid (50 lg/mL) and ampicillin (50 lg/mL) to avoid bacterial contamination. The general analysis was based on the comparison of three different nitrogen conditions. A reference condition called the sugar cane juice without the addition of ammonium sulfate, naturally containing 81.2 4.9 mg N/L, and two nitrogen supplementation conditions, one a mid-range ammonium sulfate concentration of 440 mg/L (MAS) representing 175 4.9 mg N/L, and the other with a high ammonium sulfate supplementation to 5830 mg/L (HAS) representing 1320 4.9 mg N/L (Table 1). After rehydration and centrifugation, 6 g of cells (wet weight) were inoculated in 50 mL of natural sugar cane juice prepared as described below, to result in a nal concentration of 120 g cells (wet weight)/L (ca. 109 cells/mL). Micro-fermentations were carried out in three independent biological replicates at 32 C under gentle agitation (enough to prevent cell sedimentation) for 9 h until almost complete consumption of sucrose (>99.5%). The cell density was measured by cell counting using a haemocytometer and the sugar consumption was determined every 2 h by means of a manual refractometer and expressed as Brix. Three samples of 1 mL were withdrawn at the beginning and after 9 h of fermentation and centrifuged at 5000g for 30 s. The supernatant was used for the measurement of metabolites and the cell pellets were frozen in liquid nitrogen and stored at 80 C for RNA extraction. 2.2. Gene expression analysis Total RNA was extracted from yeast cells by the phenol/chloroform method as previously described (Schmitt, Brown, & Trumpower, 1990). The quality of the extracted RNA was estimated by loading 1 ll of the sample on 1% agarose gel. The remaining RNA was kept at 80 C until used. The quantitative analysis was performed by RT-qPCR using the primers described in Table S1 (Supplementary data). The ADK1 (Oechsner, Magdolen, Zoglowek, Hcker, & Bandlow, 1988), PDA1 (Wenzel, Teunissen, & Steensma, 1995) and EFB1 (Hiraga, Suzuki, Tsuchiya, & Miyakawa, 1993) genes were used as references for gene expression analysis (Fig. S1, Supplementary data). The nucleotide gene sequences were obtained from the Saccharomyces Genome Database (SGD) and used for primer design with the aid of the on-line Genescript primer design program in advanced mode (www.genscript.com/cgibin/tools) with the following parameters: 1725 nucleotide length bases, 59 C Tm value, 70110 bp amplicon length. The primers were checked for self-hybrids, hairpins and loops by the Netprimer tool (www.premierbiosoft.com/netprimer/netprlaunch/netprlaunch.html). Only the primers with a ranking higher than 90% were selected for the next round of selection. Afterwards, the matches and specicity to target genes were checked by BLAST analysis against the S. cerevisiae whole genome. Finally, in silico PCR (http://genome.ucsc.edu/ cgi-bin/hgPcr) using S. cerevisiae whole genome was performed to check the specicity of the reaction.cDNA synthesis was performed with the ImProm-II Reverse Transcription System kit from Promega (Madison, WI), using s instrucOligo(dT)15 as primers and following the manufacturer tions. The mRNA quantication was carried out by SYBR Green PCR Master Mix kit (Applied Biosystems, Foster City, CA) using the detection system 7500 Real Time PCR system (Applied Biosystems), following the manufacturers instructions. The following conditions were required for the amplication: initial step at

2. Materials and methods 2.1. Strain and fermentation assays The industrial S. cerevisiae strain JP-1 (Silva-Filho et al., 2005) is a commercial yeast strain used for industrial bioethanol production in Brazil, as well as for cachaa fermentation. In this study, dry yeast cells (Fermol Distiller, AEB Bioqumica Latino Americana, So Jos dos Pinhais, Brazil) were rehydrated in sterile distilled water at 30% w/v for 30 min with agitation. All the micro-fermentation assays used fresh sugar cane juice provided by Still B, a manufacturer of artisanal cachaa from Igarass city, Pernambuco state, Brazil (Vilanova et al., 2009). After the sugar cane had been crushed, the extracted juice was kept ice-cold and

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703

Table 1 Composition of the sugar cane juice media used for JP-1 strain fermentation and fermentative parameters. (N = nitrogen, C = carbon, S = sucrose consumed, P = ethanol produced, Y = ethanol yield from sucrose in g/g). Type Nitrogen Supply Total N Sucrosea Initial Consumed S (mg/L) SCJ LAS HAS
a b c d

C/N Total C

Ethanola Produced P Consversion Y

(P/S)

(mg N/L) 81.21b 4.9 93.28 1235

(mg N/L) 81.2 4.9 174.52d 4.9 1317.2d 4.9

(g/L) 130.43 0.94 131.63 0.93 138.44 0.90

(g/L) 129.01 0.94 129.69 0.94 137.14 0.91

(%) 98.91 98.53 99.06

(g C/L) 54.87 55.37 58.24 675.65 317.30 44.21

(g/L) 47.98 0.21 43.37 2.81 54.09 4.03

(g/g) 0.37 0.01 0.33 0.07 0.39 0.07

NH 4 (NH 4 2 SO4 ((NH 4 2 SO4

440c 5830c

HPLC determination in this work. NH 4 present in the sugar cane juice. Concentration of added ammonium sulfate. Amount corresponding to the ammonium present naturally in the sugar cane juice plus the nitrogen supply.

50 C for 2 min and 95 C at 10 min followed by 40 cycles of 95 C for 15 s and 60 C for 1 min. To calculate the level of genomic DNA contamination, amplications were performed without reverse transcriptase. The results showed gDNA lower than 1.5% of cDNA (data not provided). The quantication of gene expression used the method of relative quantication of 2DDCt (Livak & Schmittgen, 2001). The values represent the average, at the relative mRNA level, of three independent biological replicates with at least three technical replicates each. The results were statistically evaluated by determining the difference between the averages using ANOVA (p value of <0.05) followed by Tukey test (p value of <0.05) for minimal signicant differences within each treatment. Statistical analysis showed that values of 0.2 or above as signicant differences. To measure the errors of the target genes was used the p error propagation rules: d = [(d1)2 + (d2)2], where d1 is the standard error of the target gene and d2 is the standard error for endogenous control. 2.3. Measurements of metabolites 2.3.1. Sucrose, glucose, fructose, acetate, ethanol and glycerol The determination of sugar and organic acids from fermentation supernatants was performed by HPLC using an Aminex HPX-87H+ column (300 7.8 mm) under the following conditions: temperature 40 C, solvent H2SO4 (5 mM), a ow rate of 0.5 mL/min and a dual detection system (refractometer and UV at 210 nm). The compounds were identied by considering the relative retention time and quantied on the basis of direct comparison using a standard curve. This was carried out by forming a mixture of sucrose, ethanol, glucose, fructose, acetate and glycerol (20 g/L each) in ultrapure water. The values represent the average of three independent biological replicates with at least two technical replicates each. Analysis of the data found that the error in measurement was less than 10% in all cases. 2.3.2. Esters and higher alcohols The concentration of ester in the medium was determined by GC, using a Carbowax 20 M column (30 m 0.53 mm 0.5 mm) with hydrogen as the carrier gas and ame ionisation detection (CG-Master; Ciola & Gregori Ltda, So Paulo, Brazil), under the following conditions: an injection temperature of 130 C, initial temperature of oven of 40 C (6 min) to nal temperature of 110 C (2 min) at a rate of 5 C/min, a ow rate of carrier gas of 7 mL/ min, a detector temperature of 130 C and an injected volume of 20 ll. For the determination of higher alcohols, a liquid/liquid extraction 50% (v/v) (diethyl ether with octanol 1 lg/mL as internal standard) was performed. The organic phase was extracted after mechanical agitation for one minute and centrifugation for ve minutes at 4000g. The concentration of higher alcohols in the organic phase was determined by GC, using a Poraplot Q column

(25 m 0.53 mm) with hydrogen as the carrier gas and ame ionisation detection (Hewlett Packard 5890A), under the following conditions: an injection temperature of 250 C, initial oven temperature of 115 C to nal temperature of 235 C at a rate of 12 C/min and an a nal isotherm of 10 min, a ow rate of carrier gas of 20 mL/min and an injected volume of 10 ll. All compounds were identied by their relative retention times, normalised with internal standard (octanol) and quantied by means of a standard curve made with each individual compound. This was conducted by preparing a mixture of 2-methyl-1-propanol (2-MP), 2methyl-1-butanol (2-MB), 3-methyl-1-butanol (3-MB), 3-methylbutyl acetate (3-MBA), ethyl acetate (EA) ethyl hexanoate (EH) and ethyl octanoate (EO) at 1 g/L in 30% (v/v) ethanol solution. The values represent the average of three independent biological replicates with two technical replicates. 2.3.3. Free amino nitrogen (FAN) Free amino nitrogen (ammonia plus amino acids) was determined by a spectrophotometric assay (Abernathy, Spedding, & Starcher, 2009). The calibration standard was glycine. The colour reagent used was ninhydrin 2% in 4 N ethylene glycol/sodium acetate buffer (pH 5.5). The absorbance of each reaction was measured at 575 nm against a blank containing water in place of the sample. 2.3.4. Carbon balance calculation Organic compounds produced in fermentation of sugar cane juice by JP-1 industrial yeast strain under micro-aerobic conditions were estimated from the HPLC and GC quantication and transformed into carbon atom (mM), to analyse the partition of initial carbon atoms (in the form of sucrose) in the total compounds quantied (Table 2). The residual sugar was considered in the calculation and, hence, the total percentage is related to the sucrose input. For the estimation of assimilated carbon converted to cell biomass, the elementary composition formula reported for S. cerevisiae, CH1.83O0.56N0.17 was used (Roels, 1983). The carbon dioxide produced during fermentation was calculated according to the assumption that 1 mol of carbon dioxide is formed for every mole of ethanol or acetic acid produced, and that 424 mmol carbon dioxide is generated from 4000 mmol cell biomass (100 g dry weight) (Van Dijken & Scheffers, 1986). 3. Results and discussion 3.1. Fermentation parameters for ammonium supplementation In this study, the industrial yeast strain S. cerevisiae JP-1 was used to ferment sugar cane juice supplemented with ammonium sulfate at different concentration levels under industrial-like conditions, i.e., a specically elevated concentration of sucrose, high

704 Table 2 Carbon balance of the sugar cane batch fermentations. Carbon SCJ (mM) Input Residual Sucrose Sucrose Glucose Fructose Glycerol Acetate Ethanol Succinate Biomass 2-MP 3-MB 2-MB 2-PE EO EH 3-MBA EA

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MAS (%) 100.00 1.09 2.92 16.22 6.59 0.58 45.56 0.42 22.78 0.10 0.00 0.00 0.00 0.00 0.37 0.3 0.29 0.00 97.23 (mM) 4614.60 32.60 68.23 4.53 320.10 23.48 1040.98 70.65 271.05 20.20 27.92 16.33 1882.98 122.16 15.14 1.12 941.49 61.08 4.74 0.015 0.00 0.007 0.00 0.003 0.00 0.001 0.00 10.50 0.73 12.48 0.88 53.69 3.76 0.03 0.00 4649.4 (%) 100.00 1.48 6.6 22.57 4.96 0.01 47.41 0.33 23.7 0.1 0.00 0.00 0.00 0.00 0.23 0.27 1.16 0.00 108.81

HAS (mM) 4853.34 31.55 45.46 3.97 27.44 1.03 410.85 10.09 329.07 33.03 63.34 18.30 2348.11 174.80 10.95 2.45 1174.05 87.40 4.74 0.015 0.00 0.004 0.00 0.003 0.00 0.001 0.00 31.20 2.19 9.84 0.69 7.91 0.56 0.02 0.00 4463 (%) 100.00 0.94 0.57 8.47 6.78 1.31 48.38 0.23 24.19 0.10 0.00 0.00 0.00 0.00 0.64 0.2 0.16 0.00 91.96

Products

Aromas

Total recovery

4572.53 32.95 49.90 1.19 133.58 7.38 741.64 23.38 301.44 5.15 26.68 0.36 2083.11 8.93 19.26 0.46 1041.55 4.46 4.74 0.018 0.00 0.008 0.00 0.004 0.00 0.001 0.00 16.90 1.18 13.68 0.96 13.30 0.93 0.01 0.00 4445.8

2-MP: 2-methyl-1-propanol, 2-MB: 2-methyl-1-butanol, 3-MB: 3-methyl-1-butanol, 3-MBA: 3-methylbutyl acetate, EA: ethyl acetate, EH: ethyl hexanoate, EO: ethyl octanoateoctanoate.

yeast biomass, elevated temperature, an absence of aeration and minimal agitation. The fermentation parameters are shown in Table 1. Diluted sugar cane juice used for bioethanol and cachaa elaboration contains a sucrose concentration between 130 and 140 g/L and a nitrogen source in the range of 7485 mg N/L (Table 1). In the context of cachaa production, there is a certain difculty to nd out what concentration of nitrogen in the medium would be optimal for the cell population required. The nitrogen demand will depend of several parameters, as for example the type of cells, given by the strain of yeast used, the size of inoculum, and the type of fermentation conditions employed, such as aeration and sugar concentration. Another criterion that could be useful when examining the nutritional fermentation requirement is the carbon/nitrogen ratio (C/N). For example, for the very well dened medium Yeast Peptone Dextrose (YPD) the ratio C/N is about 1.45, while in completely synthetic medium SC the C/N ratio is about 6.65. In the wine industry, Wang et al. (2003), Jimnez-Mart, Aranda, Mendes-Ferreira, Mendes-Faia, and del Olmo (2007), and Jimnez-Mart and Del Olmo (2008) dene as low nitrogen a medium with a C/N ratio of 13301640, while a high nitrogen medium possesses a C/N ratio of 260380. Moreover, in the beer context, OConnor-Cox and Ingledew (1989), and Jones and Rainbow (1966) dene as optimal nitrogen concentration a C/N ratio of 44.5 and 30 respectively. It seems that a C/N ratio below 50 would be appropriate, independently of the fermentation parameters used. In our case, supplementation of 440 mg/L ammonium sulfate (MAS), in addition to the nitrogen already present in the must would yield a nal assimilable nitrogen concentration of 174 mg/ L, resulting in a C/N ratio of 317 (Table 1), whereas a supplementation of 5830 mg/L ammonium sulfate (HAS) would yield 1320 mg assimilable nitrogen/L, producing a C/N ratio of 44, which is well above the optimal C/N proposed. As shown in Fig 1, whatsoever the initial content of nitrogen, the fermentation behaviour of the sugar cane juice showed no increase in the biomass level, even though 99.5% of the initial sucrose content was consumed (Table 1 and Fig. 1). However, there were signs of the presence of a measurable amount of glucose and in particular, fructose in the wort at the end of the process (between 9% and 24% of the initial sucrose), suggesting there had been a difference in the whole fermentation process between the different ammonium situations. Residual sugar (sucrose, glucose plus

Fig. 1. Sugar consumption (as dissolved solid content) and biomass evolution of sugar cane juice fermentation (SCJ) or sugar cane juice supplemented with 440 mg/ L (MAS) or 5830 mg/L (HAS) of ammonium sulfate by the JP-1 yeast strain.

fructose) was 21% in the SCJ condition (27.7 g/L), 32% in the MAS (42.8 g/L) and 10% in HAS condition (14.5 g/L), in relation to the initial sucrose input (Table 1 and Fig. 2). Carbon balance analysis showed that all the carbon input was recovered at the end of the fermentation, either as residual sugar or metabolite products (Table 2). The principal metabolite produced was ethanol, representing 4649% of the total sucrose, whereas the second was glycerol, corresponding to 57% of total sucrose (Table 2). Acetate only represented 0.011.4%. These results conrmed the micro-aerobics conditions used. In comparison to SCJ, ethanol yield was 12% higher in HAS while it was 9.6% lower in MAS. Also, acetate level was 2.37 times higher in HAS (Fig. 2). As summarised in Table 1 and Fig. 1, supplementation with mid-range concentration of ammonium sulfate (MAS) showed a detrimental effect on the fermentation parameters (ethanol conversion and production), whereas other parameters (ethanol, acetate and glycerol yield) were significantly increased at high ammonium supplementation, concomitantly with a better assimilation of sucrose, an increased CO2 production and a reduction of succinate. The decline in the succinate production could be a result of the reduced activity of the

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705

Fig. 2. Metabolite production after 9 h of fermentation in sugar cane juice (SCJ) or sugar cane juice supplemented with 440 mg/L (MAS) or 5830 mg/L (HAS) of ammonium sulfate by the JP-1 yeast strain. (Suc: sucrose; Gluc: glucose; Fruc: fructose; Ace: acetate; Gly: glycerol; Succ: succinate; Eth: ethanol).

citric acid cycle, due to pyruvate deviation towards ethanol production with increasing nitrogen concentration. Surprisingly, the fermentative parameters in MAS were less than those observed in the SCJ reference. Altogether, these results indicated that a C/N ratio above 50 has a stimulatory effect on the fermentation in relation to the reference conditions when an intermediate effect between SCJ and HAS was expected. 3.2. Effects of nitrogen supplementation on higher alcohol metabolism As shown in Fig. 3A, ammonium sulfate supplementation to sugar cane juice caused a measurable decrease in higher alcohol content. The most pronounced effect was observed in the levels of 3-MB (45.7%), followed by 2-PE (36.6%), 2-MB production (24.4%) and then 2-MP (14.7%) under HAS fermentation conditions as compared to those of SCJ. This nding is in contrast to that which occurs in beer fermentation, where either excessive or low amino nitrogen tends to cause elevated higher alcohol concentrations (Boulton & Quain, 2001; Szlavko, 1974), but is similar to wine production, where higher alcohols were inclined to decrease with an increase in total nitrogen concentration (Bell & Henschke, 2008; Carrau et al., 2008). In agreement with this decrease, the expression of the genes encoding decarboxylases and alcohol dehydrogenases that are involved in the synthesis of higher alcohols were downregulated with increasing ammonium sulfate in the wort, with the exception of phenylpyruvate decarboxylaseencoding ARO10 that displayed a 5-fold upregulation under HAS conditions (Fig. 3B). This result could suggest that the formation of the higher alcohols under cachaa production is pretty much linked to the function of any of the keto-acid decarboxylases PDC1, PDC5, PDC6, THI3 (PDCs) and alcohol dehydrogenases ADH3,

ADH4 and ADH5. Interestingly, in our industrial cachaa-like conditions, the increase of ARO10 expression was concomitant with the higher supply of ammonium salt. Marks, Merwe, and Vuuren (2003) also showed a relation between diammonium phosphate supplementation and the induction of expression of genes associated with higher alcohol formation. A very recent report showed the contribution of Aro10p as the key enzyme in the production of higher alcohols, suggeting ARO10 as the principal contributor to the avour-related decarboxylation reactions during the fermentation of alcoholic beverages with S. cerevisiae (Romagnoli, Luttik, Ktter, Pronk, & Daran, 2012). The evident inconsistencies between preferred nitrogen source, higher alcohol production and respective gene expression still require investigation. All ve alcohol dehydrogenase isoforms as well as the bifunctional enzyme Sfa1p are somehow involved in the formation of higher alcohols during fermentation (Dickinson, Salgado, & Hewlins, 2003). In the present analysis, only ADH6 gene expression remained unchanged in all the fermentation conditions that were tested. Because of the large number of alcohol dehydrogenase encoding genes, the existence of a complementary effect can be argued for that does not allow the measurement of the individual contribution of each of these enzymes in alcohol production. In addition, it the involvement of other enzymes with dehydrogenase capacity cannot be excluded, for instance aryl-alcohol dehydrogenase (Delneri, Gardner, & Oliver, 1999). Equally important, the expression of LEU4 gene was downregulated 2.5-fold under HAS conditions (Fig. 3B). This gene encodes the rst enzyme in the leucine pathway, which converts 2-oxoisovalerate, a common intermediate of both leucine and valine biosynthesis, into a-isopropylmalate (Oba, Nomiyama, Hirakawa, Tashiro, & Kuhara, 2005) and results in 3-MB production. The

B 10
transcripts levels (fold change)

MASr HASr

Pyruvate decarboxilases

0
RG PDC1 PDC5 PDC6

Alcohol dehydrogenases

Isoprpyl malate synthase

THI3 ARO10 ADH3 ADH4 ADH5 ADH6 SFA1

LEU4

Fig. 3. (A) Higher alcohol production by the JP-1 yeast strain after 9 h of fermentation in sugar-cane juice (SCJ) or sugar cane juice supplemented with 440 mg/L (MAS) or 5830 mg/L (HAS) of ammonium sulfate. (2-MP) 2-methyl-1-propanol, (2-MB) 2-methyl-1-butanol, (3-MB) 3-methyl-1-butanol. (B) Relative transcription prole by the JP-1 yeast strain of genes involved in higher alcohol production. MASr and HASr represent the MAS and HAS supplemented ammonium sulfate condition relative to the condition without supplementation of ammonium sulfate (SCJ). The reference genes used for normalisation data were ADK1, PDA1 and EFB1.

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B
transcripts levels (fold change)

10
MASr HASr

0
RG

Acetyl-CoA synthase Fatty acid synthase

Alcohol acetyl/acyl transferases

Esterase

ACS2

FAS1

FAS2

ATF1

ATF2

EEB1

IAH1

Fig. 4. (A) Ester production by the JP-1 yeast strain after 9 h of fermentation in sugar cane juice (SCJ) or sugar cane juice supplemented with 440 mg/L (MAS) or 5830 mg/L (HAS) of ammonium sulfate. (3-MBA) 3-methylbutyl acetate, (EA) ethyl acetate, (EH) ethyl hexanoate and (EO) ethyl octanoate. (B) Relative transcription prole by the JP-1 yeast strain for genes involved in ester production. MASr and HASr represent the medium and high supplemented ammonium sulfate samples relative to the control (SCJ). The reference genes used were ADK1, PDA1 and EFB1.

observed downregulation of LEU4 gene by high ammonium concentration may be directly related to inactivation of its transcription factor inducer Gcn4p at a high level of ammonium in the medium (Hinnebusch & Natarajan, 2002). This downregulation of the ux towards a-isopropylmalate biosynthesis, together with the downregulation of both decarboxylase and dehydrogenaseencoding genes upon nitrogen supplementation, might explain the pronounced reduction of 3-MB (45.7%) under high nitrogen supplementation (Fig. 3A). The fact that higher alcohols and succinate showed similar proles in response to ammonium addition might indicate a common pathway. One explication for the higher alcohols formation could be the auxiliary mechanism of ne setting intracellular oxidoreductive balance for regeneration of NAD+, in addition to glycerol formation (Lilly, Bauer, Styger, Lambrechts, & Pretorius, 2006; Lodolo, Kock, Axcell, & Brooks, 2008; Quain & Dufeld, 1985). Here, the reduction in concentration of higher alcohol and succinate was also concomitant with increased levels of glycerol and ethanol, which further support the notion that redox balance and fermentation performance is closely associated to nitrogen availability. 3.3. Effects of nitrogen supplementation on the metabolism of esters The production of ethyl esters was somewhat variable in response to nitrogen supply. As shown in Fig. 4A, the production of ethyl octanoate was 2-fold higher in HAS than in SCJ, while maximal levels of ethyl acetate (EA) and 3-methylbutyl acetate esters were found in MAS, and ethyl hexanoate did not show signicant change with respect to ammonium content in the wort. These results are in accordance with those of Saerens et al. (2008), who showed that higher level of nitrogen cause a stronger increase in acetate ester than in ethyl ester production. Furthermore, it has been reported that lipid synthesis may compete with ester synthesis as both are dependent on the intracellular pool of acetyl-CoA (Boulton & Quain, 2001). Under our culture conditions, and mainly under HAS, there was a signicant downregulation of genes of fatty acid synthesis (FAS1 and FAS2), together with a decrease in expression of the genes encoding alcohol acetyl transferase (ATF1 and ATF2) (Fig. 4B). This nding could account for the decrease in ethyl esters (Fig. 4A). Inversely, this behaviour could promote the acetate ester production by leaving more acetyl-CoA available, as also evidenced by increased levels of 3-methylbutyl acetate and ethyl acetate content in MAS (Fig. 4A). The downregulation observed for the ATF1 gene (Fig. 4B) under the nitrogen-supplementation oxygenlimited conditions used here was in contradiction of its upregulation by high levels of nitrogen in the medium (Verstrepen et al., 2003). Possibly, the inhibitory effect of unsaturated fatty acid pres-

ent in the sugar cane could be one of the causes in the reduction of ATF1 and ATF2 transcription. It was also found that expression of EEB1, encoding a mediumchain acyl ester synthase (Saerens et al., 2006), followed the concentration prole of ethyl octanoate (Fig. 4A). In this context, It was observed that the enzyme activity associated with overexpression of EEB1 did not result in an increase in the production of ethyl esters (Saerens et al., 2008), assigning this behaviour to the hydrolytic activity of this enzyme (Saerens et al., 2006). This might provide an alternative explanation for the absence of an increase in the nal ester level, where the balance between transferase/esterase activities is the principal factor. In this respect, the product of IAH1 was described to show an isoamyl acetate esterase activity (Fukuda et al., 2000) and its slight upregulation in HAS may also account for the decrease in acetate esters (Fig. 4A). Overall, the potential formation of esters could be reduced because of the upregulation of EEB1 and IAH1 observed in HAS, while at the same time allowing a synthesis of medium-chain acyl esters, such as ethyl octanoate and ethyl hexanoate under medium ammonium supply (Fig 4A). Altogether, the results presented here are in accordance with those observed in wine: higher must concentrations of nitrogen are associated with high concentrations of esters, particularly ethyl esters, which contribute fruity aromas, and with low higher alcohol concentrations (Bell & Henschke, 2008). 4. Conclusion This study showed that the supplementation of sugar cane juice with ammonium salt can exert a strong inuence on fermentation parameters and avour prole. Surprisingly, it was found that the mid-range addition of ammonium nitrogen had a measurable inuence on the metabolism of avours and on the expression of their related genes. In this situation, the genes of the Ehrlich pathway were repressed, resulting in a general decrease in higher alcohol production, whereas there was an increase in IAH1 encoding an esterase and in the production of acetate esters. This situation may be useful for the production of beverage with a sharpened perception of fruitiness, due to the combined action of increased 3-methylbutyl acetate and ethyl acetate. In addition, high ammonium supplementation caused an overall decrease in the transcription levels of most of the genes required for higher alcohols, with the exception of ARO10. This is accompanied by a general reduction of higher alcohols, while the cells excrete a larger quantity of ethyl octanoate, ethyl acetate and acetate in the medium. This could lead to a synergistic effect between the acidic attributes of acetate and the fruity properties of esters in the nal beverage.

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Acknowledgements This study was partly sponsored by Rgion Midi Pyrenes contract (n09005247), by grants from the French-Brazilian cooperation program CAPES-COFECUB (n99/2008) and grants from FACEPE (Ph.D. scholarship program and APQ research funding program). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2012. 10.147. References
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