Screening of native yeast from Agave duranguensis fermentation for isoamyl acetate production

357 Vol.56, n.3: pp. 357-363, May-June 2013 ISSN 1516-8913 Printed in Brazil BRAZILIAN ARCHIVES OF BIOLOGY AND TECHNOLOGY A N I N T E R N A T I O N A L J O U R N A L Screening of Native Yeast from Agave duranguensis Fermentation for Isoamyl Acetate Production Gerardo Hernández-Carbajal1, Olga Miriam Rutiaga-Quiñones1, Araceli Pérez-Silva2, Gerardo Saucedo-Castañeda3, Adriane Medeiros4, Carlos Ricardo Soccol4 and Nicolás Óscar Soto-Cruz1* 1 Instituto Tecnológico de Durango; Felipe Pesacador 1830 Ote. 34080; Durango – México. 2Instituto Tecnológico de Tuxtepec;Víctor Bravo Ahuja s/n, 68350; Tuxtepec, Oxaca. 3Universidad Autónoma Metropolitana Iztapalapa; San Rafael Atlixco 186, 09340; México. 4Universidade Federal do Paraná; 81531-990; Curitiba - PR - Brasil ABSTRACT In this work, fifty yeast strains, isolated from the spontaneous alcoholic fermentation of Agave duranguensis to produce mezcal, were tested using the double coupling system. These yeasts were from the genera Pichia, Torulaspora, Saccharomyces, Kluyveromyces, Deckera, Hanseniaspora, and Candida. P. fermentans ITD00165 was the best isoamyl acetate producer, yielding 0.38 g/L of ester after incubation for 24 h, while K. marxianus ITD00211 produced 0.32 g/L of ester. Thus P. fermentans ITD00165 could be considered as an excellent choice for use in optimization studies of the culture medium and bioreactor operating conditions to develop a process for biotechnological production of isoamyl acetate. Key words: Banana aroma, Food additive, Native yeast strains INTRODUCTION A vast array of compounds, such as alcohols, esters, fatty acids, and sulphur compounds may be responsible for the flavor of foods (Gratfield. 1988; Dubal et al. 2008; Krings 1998). Food processing can cause a weak aroma in the final product, hence, it is necessary to use the additives (Lemos et al. 2010). Most of these compounds are produced by the chemical synthesis, but a rapid shift to biosynthesis is taking place (Janssens et al. 1992) because consumers have developed a tendency to prefer the food with a "natural" label (Janssens et al. 1992; Lemos et al. 2010). Isoamyl acetate is an ester with great interest in the food industry. It has a consumption of 74,000 kg per * year due to its characteristic banana smell (Torres et al. 2009). Yeasts produce esters by esterification of alcohols with acetyl co-enzyme A (Verstrepen et al. 2003). Two genes coding for the enzyme alcohol acetyltransferase have been identified in Saccharomyces cerevisiae (Mason and Dufour. 2000). This enzyme catalyzes the reaction between acetyl co-enzyme A and alcohols. Yeasts also produce enzymes with ester hydrolase activity and the balance between these two antagonistic enzyme activities determines the final concentration of isoamyl acetate in the fermentation system (Inoue et al. 1997; Fukuda et al. 1998; Yoshimoto et al. 1999; Rojas et al. 2001). Oda (1996) developed a system for the production of esters, called a doubled coupled system (DCS), Author for correspondence: Braz. Arch. Biol. Technol. v.56 n.3: pp. 357-363, May/June 2013 358 Hernández-Carbajal, G. et al. which consisted in an immobilized microorganism between two the phases, one solid and one liquid. The hydrophilic solid surface contains the nutrients necessary for the microorganism, while the liquid phase is composed of an organic solvent in which the alcohol to be esterified is dissolved. After alcohol esterification with acetyl co-enzyme A, the resulting ester accumulates in the hydrophobic phase of the system, where it can be quantified and recovered. Like several studies on alcoholic beverage production (Valero et al., 2002; Abbas. 2006), the objective of this work was to select yeast strains with high capacity of isoamyl acetate production, using the doubled coupled system in order to take advantage of the microbial biodiversity in the region as an alternative to produce a natural banana aroma. MATERIALS AND METHODS Chemicals Isoamyl acetate (GC grade), isoamyl alcohol, decane, anhydrous sodium carbonate, potassium sodium tartrate, sodium sulphate, copper sulphate pentahydrate, sulphuric acid, ammonium molybdate, sodium arsenate heptahydrate, and glucose were supplied by Sigma-Aldrich (USA). Peptone, yeast extract, malt extract, and agar were supplied by BD Bioxon (Mexico). Yeast Strains Fifty non-Saccharomyces strains belonging to the genera Candida, Hanseniaspora, Kluyveromyces, Pichia, Torulaspora, and Dekkera, isolated from the spontaneous alcoholic fermentation of Agave duranguensis, were obtained from the Microbial Biotechnology Lab’s Culture Collection at the Durango Institute of Technology. Screening procedure The synthesis of isoamyl acetate was carried out following the protocol described by Oda (1996). Two hundred microliters from 24 h cultures in GPYM medium were spread onto Petri dishes with solid GYMP medium and incubated at 30 °C for 24 h. After incubation, 8.0 mL of a 1% (v/v) isoamyl alcohol solution in decane was placed onto the agar plate and incubated at 30 °C for 24 h with shaking (100 rpm). A negative control was subjected to the same process by adding an isoamyl aceteate in decane solution (0.3 g/L) onto Petri dishes without microorganism. Aliquots of the decane solution were taken after incubation for 6, 12, and 24 h for analysis by gas chromatography (GC). The GYMP medium contained (g/L): 40 glucose, 5 peptone, 3 yeast extract, 3 malt extract, and 1 magnesium sulfate heptahydrate. The pH of the medium was adjusted to 6.0. Isoamyl acetate and isoamyl alcohol concentrations were determined using standard solutions. Measurements reported are the average of three independent plate cultures. Statistical comparisons were made using Analysis of Variance (ANOVA) and the Fisher Least Significant Difference (LSD) post-hoc test with the program Statistica version 7.0 (StatSoft, USA). The concentration of isoamyl acetate was quantified on an Agilent Technologies Network System Gas Chromatograph 6890N (Agilent Technologies, USA) equipped with a flame ionization detector (FID) and an HP-Innowax column (Hewlett-Packard, USA) (length, 30 m; inside diameter, 0.25 mm; film thickness, 0.25 mm). The temperatures of the injector block and detector were 220 and 300 °C, respectively. The oven temperature was programmed as follows: equilibrated at 80°C for 10 min, 80 to 150 °C at 1.5 °C/min, heated to 170 °C at 4°C/min, heated to 250 °C at 20 °C/min, and maintained at 250 °C for 2 min. Liquid culture for acetate ester formation To measure the ester formation, liquid cultures were grown in 125 mL flasks containing 50 mL of sterilized GPYM medium at pH 6.0. Flasks were incubated at 28 °C with shaking (120 rpm). Aroma compounds in the culture were measured by the headspace gas chromatography. Isoamyl alcohol (1%), sterilized with a 0.2 µm filter, was added to flasks at 0 and 12 h of fermentation to look at its effect on ester production. Yeast cell counts were determined by a Neub (...truncated)