STUDY OF INCREASING THE PRODUCTION OF VOLATILE FLAVOR COMPOUNDS BY THE YEAST Kluyveromyces marxianus THROUGH OPTIMIZATION OF CARBON AND NITROGEN SOURCES

Food and Health, 4(2), 112-123 (2018) • DOI: 10.3153/FH18011 E-ISSN: 2602-2834 Original Article/Full Paper STUDY OF INCREASING THE PRODUCTION OF VOLATILE FLAVOR COMPOUNDS BY THE YEAST Kluyveromyces marxianus THROUGH OPTIMIZATION OF CARBON AND NITROGEN SOURCES Müge İşleten Hoşoğlu Cite this article as: İşleten Hoşoğlu, M. (2018). Study of Increasing the Production of Volatile Flavor Compounds by the Yeast Kluyveromyces marxianus Through Optimization of Carbon and Nitrogen Sources. Food and Health, 4(2), 112-123. DOI: 10.3153/FH18011 Canakkale Onsekiz Mart University, Food Engineering Department, Terzioglu Campus, Canakkale, Turkey ABSTRACT The regulation of growth and the production of flavor compounds by Kluyveromyces marxianus were accomplished according to the nutritional requirements (yeast extract, ammonium sulphate and glucose concentrations) by using Response Surface Methodology experiments. Results proved that increasing both initial yeast extract (YE) and glucose concentrations in the fermentation medium favored both the growth and production of fusel alcohols. The major fusel alcohol (isoamyl alcohol) and the acetate ester compound (ethyl acetate) produced in all flasks were determined in the concentration range 1299-3996 µg L-1 and 1558 to 3122 µg L-1, respectively. In a scale-up attempt, productions were accomplished in a 5 L stirred tank bioreactor. The highest productivity values for major volatile flavor compounds, i.e., ethyl acetate (fruity), isoamyl alcohol (banana), 2-phenylethyl acetate (floral) were obtained during the exponential growth of the yeast in a 5 L stirred tank bioreactor. Additionally, the descriptive sensory terms “sourdough”, “flower” and “sweet aromatic” were characteristics for volatile compounds produced by K. marxianus. This work also demonstrated high product losses due to the stripping effect when the production scaled up from flasks to bioreactor. Keywords: Kluyveromyces marxianus, Natural flavor compounds, Optimization, Bioreactor, Sensory evaluation Submitted: 30.05.2017 Accepted: 11.11.2017 Published online: 22.01.2018 Correspondence: Müge İŞLETEN HOŞOĞLU E-mail: ©Copyright 2018 by ScientificWebJournals Available online at www.scientificwebjournals.com 112 Food and Health, 4(2), 112-123 (2018) • DOI: 10.3153/FH18011 E-ISSN: 2602-2834 Original Article/Full Paper Introduction Due to an increasing preference of the consumers for natural food additives and other compounds of biological origin, there has been increasing trend towards the production of flavor compounds by biotechnological process (Janssens et al. 1992; Medeiros et al. 2000; Mantzouridou et al. 2013; Berger, 2015). Although several bacteria, yeasts and fungi have been reported for the production of flavor compounds, a few species of yeasts and fungi have generally been preferred, and only a few of these find industrial application due to their GRAS (generally recognized as safe) status. Among the different producers, food grade yeast Kluyveromyces marxianus has been pointed out as a promising organism for the production of natural flavor compounds such as fruit esters, carboxylic acids, ketones, furans, alcohols, monoterpene alcohols, and isoamyl acetate in liquid fermentation (Medeiros et al. 2000; Gethins et al. 2015; Morrissey et al. 2015). Because of its ability to grow on a broad variety of substrates, at higher temperatures and rapid growth, many efforts have been made with this yeast like production of enzymes (Panesar, 2008), single cell protein (Aggelopoulos et al. 2014), reduction of lactose content in food products (Manera et al., 2008). With regard to the production of flavor compounds using K. marxianus, most progress has been made specifically with the production of 2-phenylethanol (2-PE) (Etschmann et al. 2003; Etschmann et al. 2004), ethyl acetate (Urit et al. 2003a; Urit et al. 2003b) and the production of total volatile compounds by using inexpensive waste-stream medium as the growth medium (Medeiros et al. 2000; Wilkowska et al, 2014; Guneser et al. 2015). However, industrial production of sufficient quantities of yeast as starter cultures implies characterization of yeast physiology and nutritional requirements. Therefore, it is important to assess what kind of flavor compounds are synthesized by K. marxianus when growing on defined and/or semidefined culture medium rather than growing on waste stream (Gethins et al. 2015). Medium composition especially carbon and nitrogen sources, is one of the critical factor for both growth and production flavor compounds by yeasts (Fabre et al. 1998; Gethins et al. 2015; Löser et al. 2015). Hence, to obtain high biomass and metabolite productivities, it is important to find appropriate types of nutrients and their optimum concentrations in growth medium of the yeast (Manera et al. 2008; Fonseca et al. 2013; Yilmaztekin et al. 2013). Additionally, reducing the cost of culture medium by optimizing its composition is the basic research for industrial applications (Etschmann et al. 2004; Manera et al. 2008). Etschmann et al. (2004) optimized the growth medium of K. marxianus CBS 600 for production of 2-PE. In a recent study, it was also revealed that nitrogen and carbon source had pronounced effects on production of volatile metabolites by four strains of K. marxianus (Gethins et al. 2015). It was concluded that, nitrogen source and carbon source had pronounced effects on production of volatile metabolites. However, there was no information about the specific nutritional requirements and their optimum concentrations in the medium for maximizing the growth and production of each volatile flavor compounds by K. marxianus. Response surface methodology (RSM) is a collection of mathematical and statistical techniques useful for designing experiments, establishing models, and analyzing the effects of several independent factors. The main advantage of RSM is the reduced number of experimental trials needed to evaluate multiple factors. Also, study of the individual and interactive effects of these factors will be helpful in efforts to find the target value. Hence, RSM provides an effective tool for investigating the aspects affecting desired response if there are many factors and interactions in the experiment (Garrido-Vidal et al. 2003; Elibol, 2004). The aims of this study were two-fold: first, on the basis of optimization of the growth medium, the present author intended to better understand the effect of carbon (glucose) and different nitrogen substances (yeast extract and ammonium sulphate) and to find their optimum medium composition for increasing the growth and the production volatile flavor compounds by Kluyveromyces marxianus NRRL YB-6373; the second aim was to determine the changes in the production of flavor compounds during the yeast growth in 5 L bioreactor in the optimized culture medium. Materials and Methods Microorganism and Inoculum Preparation The Kluyveromyces marxianus NRRL YB-637 (...truncated)